Fluorescent Methods and Materials for Directed Biomarker Signal Amplification

ABSTRACT

Methods and compositions are provided that include a multichromophore and/or multichromophore complex for identifying a target biomolecule. A sensor biomolecule, for example, an antibody can be covalently linked to the multichromophore. Additionally, a signaling chromophore can be covalently linked to the multichromophore. The arrangement is such that the signaling chromophore is capable of receiving energy from the multichromophore upon excitation of the multichromophore. Since the sensor biomolecule is capable of interacting with the target biomolecule, the multichromophore and/or multichromophore complex can provide enhanced detection signals for a target biomolecule.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional Application No.60/828.615, filed Oct. 6, 2006, which application is incorporated hereinby reference.

BACKGROUND OF THE INVENTION

fluorescent hybridization probes have developed into an important toolin the sequence-specific detection of DNA and RNA. The signals generatedby the appended fluorescent labels (or dyes) can be monitored in realtime, and provide simple, rapid, and robust methods for the detection ofbiological targets and events. Utility has been seen in applicationsranging from micro arrays and real time PCR to fluorescence in situhybridization (FISH).

Recent work in the area of muitichromophores, particularly regardingconjugated polymers (CPs) has highlighted the potential these materialshave in significantly improving the detection sensitivity of suchmethods (Liu and Bazan, Chem. Mater., 2004). The light harvestingstructures of these materials can be made water soluble and adapted toamplify the fluorescent output of various probe labels (See U.S. patentapplication Ser. No. 10/600,286, tiled Jun. 20, 2003 and Gaylord,Heeger, and Bazan, Proc. Natl., Acad. Sci,, 2002, both of which areincorporated herein by reference in their entirety).

In particular, cationic CPs have shown strong affinity for oppositelycharged nucleic acids, ensuring the distances required to transferenergy from a photo-excited polymer (a light harvesting donor) to afluorescently labeled probe/target pair. The light output can beincreased by 75-fold relative to the directly excited dye alone (Liu andBazan, J. Am. Chem. Soc., 2005). The signal amplification adds a varietyof benefits in both homogeneous and heterogeneous detection formats.

Results such as these indicate CPs to be highly promising in the fieldof nucleic acid diagnostics, particularly where sample quantities arescarce. However, there exist methods for the amplification (orreplication) of nucleic acid targets, i.e., PCR. Comparatively, in thefield of protein recognition, there are no such simple methods foramplifying the targeted materials. As such, signal enhancement arisingfrom CP application is of high consequence in this area.

Dye-labeled antibodies are regularly used for the detection of proteintargets in applications such as immunohistochemistry, protein arrays,ELISA tests, and flow cytometry. Integrating CP materials into suchmethodologies promise to provide a dramatic boost in the performance ofsuch assays, enabling detection levels previously unattainable withconventional dyes.

SUMMARY OF THE INVENTION

In general, in one aspect, an assay method includes providing a samplethat is suspected of containing a target biomolecule, providing a sensorconjugated to a signaling chromophore and capable of interacting withthe target biomolecule, providing a conjugated polymer including but notlimited to

wherein the polymer electrostatically interacts with the sensor and uponexcitation is capable of transferring energy to the sensor signalingchromophore, contacting the sample with the sensor and themultichromophore in a solution under conditions in which the sensor canbind to the target biomolecule if present, applying a light source tothe sample that can excite the multichromophore, and detecting whetherlight is emitted from the signaling chromophore.

In one embodiment the R group is sulfonate. In another einbodiment thesensor is a biomolecule, for example protein, nucleic acid or anantibody.

In another embodiment the sensor can include a plurality of sensorsconjugated to a plurality of signaling chromophores, wherein at leasttwo of the plurality of chromophores emit different wavelengths of lightupon energy transfer from the multichromophore.

In general, in another aspect a multichromophore complex including amultichromophore coupled to at least one biomolecule is provided. Thebiomolecule can include but is not limited to a sensor biomolecule, abioconjugate and a target biomolecule. The multichromophore of thecomplex is further coupled to a signaling chromophore and includes thefollowing structure:

wherein CP1, CP2, CP3, and CP4 are optionally substituted conjugatedpolymer segments or oligomeric structures, that are the same ordifferent from one another. In one embodiment the conjugated polymer isa cationic conjugated polymer. In another embodiment the conjugatedpolymer is an anionic conjugated polymer. In a further embodiment theconjugated polymer is a charge-neutral conjugated polymer. In oneembodiment CP₁, CP₂, CP₃, and CP₄ are aromatic repeat units, selectedfrom the group consisting of benzene, naphthalene, anthracene, fluorene,thiophene, furan, pyridine, and oxadiazole, each optionally substituted,and wherein CP₃ and CP₄ can contain one or more unique bioconjugationsites, linked by a linker.

In an alternative embodiment multichromophore includes bioconjugationsites including but not limited to maleimide, thiol, succimidylester(NHS ester), amine, azide chemistry, carboxy/EDC(1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide Hydrochloride, Sulfo-SMCC(Sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate),amine/BMPH (N-[β-Maleimidopropionic acid]hydrazide.TFA), and Sulfo-SBEDSulfosuccinimidyl[2-6-(biotinamido)-2-(p-azidobenzamido)-hexanoamido]-ethyl-1,3′-dithiopropionate.

The multichromophore of the complex has the structure:

wherein R¹ is a solubilizing group including but not limited to ethyleneglycol oligomers, ethylene glycol polymers, ω-ammonium alkoxy sails, andω-sulfonate, alkoxy salts.

Alternatively, in another embodiment, the multichromophore of thecomplex has the structure:

wherein R¹ is a solubilizing group selected from the group consisting ofethylene glycol oligomers, ethylene glycol polymers, ω-ammonium alkoxysalts, and ω-sulfonate alkoxy salts. In particular embodiments the 1,and 2 can include a-g linking groups having the structure:

Additionally, 3 can be group h having the structure:

In another embodiment multichromophore of the complex can have thestructure:

wherein R¹ is a solubilizing group including but not limited to ethyleneglycol oligomers, ethylene glycol polymers, ω-ammonium alkoxy salts, andω-sulfonate alkoxy salts.

In still another embodiment multichromophore of the complex can have thestructure:

wherein R¹ is a solubilizing group selected from the group consisting ofethylene glycol oligomers, ethylene glycol polymers, ω-ammonium alkoxysalts, and ω-sulfonate alkoxy salts.

In yet another embodiment multichromophore of the complex can have thestructure:

wherein R¹ is a solubilizing group, including ethylene glycol oligomers,ethylene glycol polymers, ω-ammonium alkoxy sails, and ω-sulfonatealkoxy salts.

In general, in another aspect a multichromophore complex for identifyinga target biomolecule is provided that includes a multichromophore, asensor biomolecule covalently linked to the multichromophore, asignaling chromophore covalently linked to the multichromophore, whereinthe signaling chromophore is capable of receiving energy from themultichromophore upon excitation of the multichromophore and the sensorbiomolecule is capable of interacting with the target biomolecule. Inone embodiment both the signaling chromophore and the sensor biomoleculeare covalently linked to the multichromophore through a plurality oflinkers. In an alternative embodiment both the signaling chromophore andthe sensor biomolecule are covalently linked to the multichromophorethrough a tri-functionalized linker that covalently binds themultichromophore, the signaling chromophore and the sensor biomolecule.

In one embodiment the linker has a linking chemistry including but notlimited to maleimide/thiol, succimidylester (NHS ester)/amine, azidechemistry, carboxy/EDC (1-Ethyl-3-[3-dimethylaminopropyl]carbodiimideHydrochloride)/amine, amine/Sulfo-SMCC (Sulfosuccinimidyl4-[N-maleimidomethyl]cyclohexane-1-carboxylate)/thiol, and amine/BMPH(N-[β-Maleimidopropionic acid]hydrazide.TFA)/thiol. In a particularembodiment the multichromophore is a conjugated polymer, for example apolycationic conjugated polymer.

In general, in another aspect an assay method provided includes thesteps of providing a sample that is suspected of containing a targetbiomolecule, providing a multichromophore complex comprising amultichromophore, a covalently linked signaling chromophore and acovalently linked sensor biomolecule, wherein the signaling chromophoreis capable of receiving energy from the multichromophore upon excitationof the multichromophore and the sensor biomolecule is capable ofinteracting with the target biomolecule, contacting the sample with themultichromophore complex in a solution under conditions in which thesensor biomolecule can bind to the target biomolecule if present,applying a light source to the sample that can excite themultichromophore, and detecting whether light is emitted from thesignaling chromophore. In a particular embodiment the multichromophoreis a conjugated polymer, for example a polycationic conjugated polymer.

In general, in another aspect an assay method is provided including thesteps of providing a sample that is suspected of containing a targetbiomolecule, providing a first bioconjugate conjugated to a signalingchromophore and capable of interacting with the target biomolecule,providing a second bioconjugate conjugated to a multichromophore,wherein the chromophore includes the structure

wherein CP1, CP2, CP3, and CP4 are optionally substituted conjugatedpolymer segments or oligomer structures, that are the same or differentfrom one another, wherein the second bioconjugate can bind to the firstbioconjugate and wherein upon such binding excitation of themultichromophore is capable of transferring energy to the signalingchromophore, contacting the sample with the first bioconjugate in asolution under conditions in which the first bioconjugate can bind tothe target biomolecule if present, contacting the solution with thesecond bioconjugate, applying a light source to the sample that canexcite the multichromophore, and detecting whether light is emitted fromthe signaling chromophore. In one embodiment CP₁, CP₂, CP₃, and CR₂ arearomatic repeat units, including; but not limited to benzene,naphthalene, anthracene, fluorene, thiophene, furan, pyridine, andoxadiazole, each optionally substituted, and wherein CP₃ and C⁴, cancontain one or more unique bioconjugation sites, linked by a linker. Ina particular embodiment the multichromophore is a conjugated polymer,for example a polycationic conjugated polymer, an anionic conjugatedpolymer and/or a charge-neutral conjugated polymer.

In a related embodiment the multichromophore has bioconjugation sitesincluding but not limited to maleimide, thiol, succimidylester (NHSester), amine, azide chemistry, carboxy/EDC(1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide Hydrochloride, Sulfo-SMCC(Sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate),amine/BMPH (N-[β-Maleimidopropionic acid]hydrazide.TFA), and Sulfo-SBEDSulfosuccinimidyl[2-6-(biotinamido)-2-(p-azidobenzamido)-hexanoamido]-ethyl-1,3′-dithiopropionate.

In a particular embodiment the multichromophore has the structure:

wherein R¹ is a solubilizing group selected from the group consisting ofethylene glycol oligomers, ethylene glycol polymers, ω-ammonium alkoxysalts, and ω-sulfonate alkoxy salts.

In yet another embodiment the multichramophore has the stricture:

wherein R¹ is a solubilizing group selected from the group consisting ofethylene glycol oligomers, ethylene glycol polymers, ω-ammonium alkoxysalts, and ω-sulfonate alkoxy salts.

In a further embodiment 1, and 2 can include one or more a-g linkinggroups having the structure:

In one embodiment 3 is group h and as the structure:

In another embodiment multichromophore, of the complex can have thestructure:

wherein R¹ is a solubilizing group including but not limited to ethyleneglycol oligomers, ethylene glycol polymers, ω-ammonium alkoxy salts, andω-sulfonate alkoxy salts.

In yet another embodiment multichromophore of the complex can have thestructure:

wherein R¹ is a solubilizing group including but not limited to ethyleneglycol oligomers, ethylene glycol polymers, ω-ammonium alkoxy salts, andω-sulfonate alkoxy salts.

In another embodiment multichromophore of the complex can have thestructure:

wherein R¹ is a solubilizing group, including but not limited toethylene glycol oligomers, ethylene glycol polymers, ω-ammonium alkoxysalts, and ω-sulfonate alkoxy salts. In a particular embodiment themultichromophore is a conjugated polymer, for example a polycationicconjugated polymer, an anionic conjugated polymer and/or acharge-neutral conjugated polymer.

In one embodiment at least one of the first and second bioconjugate isan antibody. In a particular embodiment the first and secondbioconjugates are antibodies.

In general, in another aspect an assay method provided includes thesteps of providing a sample that is suspected of containing a targetbiomolecule, providing a multichromophore, comprising a covalentlylinked first bioconjugate, providing a sensor biomolecule, complexcomprising a sensor biomolecule capable of interacting with the targetmolecule, a signaling chromophore, and a covalently linked secondbioconjugate capable of binding with the first bioconjugate, whereinupon such binding excitation of the multichromophore is capable oftransferring energy to the signaling chromophore contacting the samplewith the sensor biomolecule complex in a solution under conditions inwhich the sensor biomolecule can bind to the target biomolecule ifpresent, contacting the solution with the multichromophore, applying alight source to the sample that can excite the multichromophore, anddetecting whether light is emitted from the signaling chromophore. In aparticular embodiment the multichromophore is a conjugated polymer, forexample a polycationic conjugated polymer, an anionic conjugated polymerand/or a charge-neutral conjugated polymer.

In one embodiment the first and second bioconjugates include but are notlimited to a protein, an antibody and a nucleic acid. In a relatedembodiment the first bioconjugate is streptavidin or biotin, the sensorbiomolecule is an antibody, and the second bioconjugate is biotin wherethe first bioconjugate is streptavidin or biotin where the firstbioconjugate is streptavidin. In another embodiment the firstbioconjugate is streptavidin or biotin, the sensor biomolecule is anucleic acid, and the second bioconjugate is biotin where the firstbioconjugate is streptavidin or biotin where the first bioconjugate isstreptavidin.

In general, in another aspect a biorecognition complex for identifying abiomolecule is provided. The complex can include a bioconjugate, asignaling chromophore covalently linked to the bioconjugate, amultichromophore covalently linked to the bioconjugate, whereinexcitation of the multichromophore is capable of transferring energy tothe signaling chromophore.

In one embodiment the bioconjugate can include but is not limited to anantibody or streptavidin. In a particular embodiment themultichromophore is a conjugated polymer, for example a polycationicconjugated polymer, an anionic conjugated polymer and/or acharge-neutral conjugated polymer.

In general, in one aspect an assay method is provided including thesteps of providing a sample that is suspected of containing a targetbiomolecule, providing a biorecognition complex comprising abioconjugate, a signaling chromophore covalently linked to thebioconjugate and a multichromophore covalently linked to thebioconjugate, wherein excitation of the multichromophore is capable oftransferring energy to the signaling chromophore, contacting the samplewith the biorecognition complex in a solution under conditions in whichthe bioconjugate can bind to the target biomolecule or atarget-associated biomolecule if present, applying a light source to thesolution that can excite the multichromophore, and detecting whetherlight is emitted from the signaling chromophore. In a particularembodiment the multichromophore is a conjugated polymer, for example apolycationic conjugated polymer, an anionic conjugated polymer and/or acharge-neutral conjugated polymer.

In general in another aspect, a biorecognition complex for identifying atarget biomolecule is provided that includes a bioconjugate, amultichromophore covalently linked to the bioconjugate, and a signalingchromophore covalently linked to the multichromophore, whereinexcitation of the multichromophore is capable of transferring energy tothe signaling chromophore. In one embodiment the bioconjugate is anantibody. In another embodiment the bioconjugate is streptavidin. In aparticular embodiment the multichromophore is a conjugated polymer, forexample a poly at conjugated polymer, an anionic conjugated polymerand/or a charge-neutral conjugated polymer.

In general in another aspect an assay method is provided including thesteps of providing a sample that is suspected of containing a targetbiomolecule, providing a biorecognition complex comprising bioconjugatecomplex comprising a bioconjugate, a multichromophore covalently linkedto the bioconjugate, and a signaling chromophore covalently linked tothe multichromophore, wherein excitation of the multichromophore iscapable of transferring energy to the signaling chromophore, contactingthe sample with the biorecognition complex in a solution underconditions in which the bioconjugate can bind to the target biomoleculeor a target-associated biomolecule if present, applying a light sourceto the solution that can excite the multichromophore, and detectingwhether light is emitted from the signaling chromophore. In a particularembodiment the multichromophore is a conjugated polymer, for example apolycationic conjugated polymer, an anionic conjugated polymer and/or acharge-neutral conjugated polymer.

In another aspect methods are provided as in any of a number of themethods disclosed herein wherein expression of a gene is detected upondetection of the target biomolecule.

In another aspect methods are provided as in any of a number of themethods disclosed herein wherein detection of the target biomoleculeprovides a result used to diagnose a disease state of a patient. In oneembodiment the method of diagnosing a disease includes the steps ofreviewing or analyzing data relating to the presence of a targetbiomolecule in a sample; and providing a conclusion to a patient, ahealth care provider or a health care manager, the conclusion beingbased on the review or analysis of data regarding a disease diagnosis.In a related embodiment providing a conclusion includes transmission ofthe data over a network.

In general, in another aspect kits for identifying a target biomoleculeare provided. In one embodiment a kit includes a multichromophore, asensor biomolecule covalently linked to the multichromophore, asignaling chromophore covalently linked to the multichromophore, whereinthe signaling chromophore is capable of receiving energy from themultichromophore upon excitation of the multichromophore and the sensorbiomolecule is capable of interacting with the target biomolecule. In aparticular embodiment the kit further includes a substrate.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1. Schematic of electrostatic binding, of a multichromophore, inone embodiment of the invention.

FIG. 2. Plot of direct excitation of a FITC-labeled antibodyillustrating amplified dye emission (left) and a schematic of thestructure of a multichromophore of one embodiment of the invention(right).

FIG. 3. Plot of direct excitation of a Cy3-labeled antibody illustratingamplified dye emission (left) and a schematic of the structure of amultichromophore of one embodiment of the invention (right).

FIG. 4. Schematic of a bioconjugated multichromophore of one embodimentof the invention.

FIG. 5. Schematic of a multichromophore conjugated to an antibody (left)or a dye (right).

FIG. 6. Schematic of a multichromophore conjugated to a secondaryantibody binding to a primary antibody labeled with dye.

FIG. 7. Schematic of a multichromophore conjugated to streptavidin forbindin and biotin-labeled primary antibody (left) or a dye- andbiotin-labeled nucleic acid (right).

FIG. 8. Schematic of multichromophore conjugated to an antibody that isconjugated to a dye (left) and a multichromophore conjugated tostreptavidin that is conjugated to a dye (right).

FIG. 9. Schematic of the structure of a multichromophore of oneembodiment of the invention.

FIG. 10. Schematic of a monomer having a bioconjugation site of oneembodiment of the invention.

FIG. 11. Schematic of a synthetic route to a monomer of one embodimentof the invention.

FIG. 12. Schematic of a synthetic route to a monomer of anotherembodiment of the invention.

FIG. 13. Schematic of a multichromophore bioconjugated to a dye and abiomolecule via linkers (left) or via a tri-functionalized linker(right).

FIG. 14. Schematic of a multichromophore conjugated to both a dye and anantibody (left) and a multichromophore conjugated to both a dye and aprotein (right).

FIG. 15. Plot of single and multiple detection of DNA probes labeledwith two dyes.

FIG. 16. Schematic of a multichromophore linked to a dye and a secondaryantibody specific for a primary antibody targeting a protein.

FIG. 17. Schematic of a multichromophore linked to a dye andstreptavidin for binding with biotin on a secondary antibody. Thesecondary antibody is shown as specific for a primary antibody thattargets a protein.

FIG. 18. Schematic of the structure of a conjugated polymermultichromophore of one embodiment of the invention.

FIG. 19. Schematic of the structure of conjugated polymermultichromophores of additional embodiments of the invention.

FIG. 20. Schematic of the structure of various aromatic units ofembodiments of the invention.

FIG. 21. Schematic of the structure of conjugated polymermultichromophores having maleimide bioconjugation sites.

FIG. 22. Block diagram showing a representative example logic device.

FIG. 23. Block diagram showing a representative example of a kit.

FIG. 24. Plot of an infrared (IR) spectroscopic analysis of anembodiment of the invention.

FIG. 25. Plot of the optical spectra of an embodiment of the invention.

FIG. 26. Plot of the fluorescence spectra of an embodiment of theinvention.

FIG. 27A. Schematic of the structure relating to biotinylation of oneembodiment of the invention.

FIG. 27B. Schematic of a biotin-avidin binding assay of the invention.

FIG. 27C. Plot of the fluorescence spectra relating to a biotin-avidinbinding assay for one embodiment of the invention.

FIG. 28. Plot of an infrared (IR) spectroscopic analysis of anotherembodiment of the invention.

FIG. 29. Plot of the optical spectra of another embodiment of theinvention.

FIG. 30A. Schematic of a control polymer structure.

FIG. 30B. Schematic of an experimental polymer structure relating to oneembodiment of the invention.

FIG. 30C. Plot of the fluorescence spectra relating to said control andan experimental polymer.

FIG. 31A. Schematics of control polymer and experimental polymerstructures relating to one embodiment of the invention.

FIG. 31B. Schematic of a fluorescence assay relating to control andexperimental polymers.

FIG. 32A. Plot of the fluorescence spectra for one embodiment of apolymer of the invention.

FIG. 32B. Plot showing corrected values for fluorescence spectra of oneembodiment of the invention.

FIG. 33A. Plot of the fluorescence signal intensity for a polymer of oneembodiment of the invention.

FIG. 33B. Plot of signal amplification relating to a polymer of oneembodiment of the invention.

FIG. 34. Plot of the optical spectra of a further embodiment of theinvention.

FIG. 35A. Schematic of a biotin-avidin binding assay of the invention.

FIG. 35B. Plot of fluorescein emission for one embodiment of theinvention.

FIG. 36A. Schematic of a polymer structure relating to one embodiment ofthe invention.

FIG. 36B. Schematic of a fluorescence assay relating to a polymer of oneembodiment of the invention.

FIG. 36C. Plot of the fluorescence spectra for one embodiment of apolymer of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Although charged multichromophore structures and basic electrostaticinteractions can be effective in the amplification of dye labeledantibodies, more directed methods of multichromophore association canensure lower backgrounds and improved signaling. The multichromophorematerials can be directly conjugated (covalently linked) to antibodiesand/or dyes providing added control (multichromophore-dye distances) inthe assay. Essentially, the signaling dye is closely coupled with theamplifying polymer. Furthermore, the conjugation of multichromophores isnot limited to dyes or antibodies; rather, the mill: chromophores can beconjugated to any variety of biomolecules, including proteins (such asavidin/streptavidin), nucleic acids, affinity ligands, sugars, lipids,peptides, and substrates for enzymes. These formats are applicable to awide variety of applications such as DNA microarrays, FISH assays, PCRassays, and also include the protein-based detection applicationsdescribed above. The properties of the polymer materials further allowfor the amplification of more than one dye using a single excitationwavelength (laser, filter, etc), This enables simultaneous detection ofmultiple targets (multiplexing). Further details relating tomultichromophores and their uses are disclosed the following, each ofwhich is incorporated herein by reference: U.S. patent application Ser.No. 11/329,495, filed Jan. 10, 2006, published as US 2006-0183140 A1;U.S. patent application Ser. No. 11/329,861, filed Jan. 10, 2006,published as US 2006-0216734 A1; U.S. patent application Ser. No.11/344,942, filed Jan. 31, 2006, published as US 2006-0204984 A1; U.S.patent application Ser. No. 10/648,945, filed Aug. 26, 2003, publishedas US 2004-0142344 A1; U.S. patent application Ser. No. 10/600,286,filed Jun. 20, 2003, published as US 2004-0219556 A1; U.S. patentapplication Ser. No. 10/666,333, filed Sep. 17, 2003, published as US2005-0059168 A1; and U.S. patent application Ser. No. 10/779,412, filedFeb. 13, 2004, published as US 2005-0003386 A1.

Before the present invention is described in further detail, it is to beunderstood that this invention is not limited to the particularmethodology, devices, solutions or apparatuses described, as suchmethods, devices, solutions or apparatuses can, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the present invention.

Use of the singular forms “a,” “an,” and “the” include plural referencesunless the context clearly dictates otherwise. Thus, for example,reference to “an aggregation sensor” includes a plurality of aggregationsensors, reference to “a probe” includes a plurality of probes, and thelike. Additionally, use of specific plural references, such as “two,”“three,” etc., read on larger numbers of the same subject less thecontext clearly dictates otherwise.

Terms such as “connected,” “attached,” “conjugated” and “linked” areused interchangeably herein and encompass direct as well as indirectconnection, attachment, linkage or conjugation unless the contextclearly dictates otherwise; in one example, the phrase “conjugatedpolymer” is used in accordance with its ordinary meaning in the art andrefers to a polymer containing an extended series of unsaturated bonds,and that context dictates that the term “conjugated” should beinterpreted as something more than simply a direct or indirectconnection, attachment or linkage.

Where a range of values is recited, it is to be understood that eachintervening integer value, and each fraction thereof, between therecited upper and lower limits of that range is also specificallydisclosed, along with each subrange between such values. The upper andlower limits of any range can independently be included in or excludedfrom the range, and each range where either, neither or both limits areincluded is also encompassed within the invention. Where a value beingdiscussed has inherent limits, for example where a component can bepresent at a concentration of from 0 to 100%, or where the pH of anaqueous solution can range from 1 to 14, those inherent limits arespecifically disclosed. Where a value is explicitly recited, it is to beunderstood that values which are about the same quantity or amount asthe recited value are also within the scope of the invention, as areranges based thereon. Where a combination is disclosed, eachsubcombination of the elements of that combination is also specificallydisclosed and is within the scope of the invention. Conversely, wheredifferent elements or groups of elements are disclosed, combinationsthereof are also disclosed. Where any element of an invention isdisclosed as having a plurality of alternatives, examples of thatinvention in which each alternative is excluded singly or in anycombination with the other alternatives are also hereby disclosed; morethan one element of an invention can have such exclusions, and allcombinations of elements having such exclusions are hereby disclosed.

Unless defined otherwise or the context clearly dictates otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. Although any methods and materials similar orequivalent to those described herein can be used in the practice ortesting of the invention, the preferred methods and materials are nowdescribed.

All publications mentioned herein are hereby incorporated by referencefor the purpose of disclosing and describing the particular materialsand methodologies for which the reference was cited. The publicationsdiscussed herein are provided solely for their disclosure prior to thefiling date of the present application. Nothing herein is to beconstrued as an admission that the invention is not entitled to antedatesuch disclosure by virtue of prior invention.

Definitions

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

“Alkyl” refers to a branched, unbranched or cyclic saturated hydrocarbongroup of 1 to 24 carbon atoms optionally substituted at one or morepositions, and includes polycyclic compounds. Examples of alkyl groupsinclude optionally substituted methyl, ethyl, n-propyl, isopropyl,n-butyl, s-butyl, t-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl,n-heptyl, n-octyl, n-decyl, hexyloctyl, tetradecyl, hexadecyl, eicosyl,tetracosyl and the like, as well as cycloalkyl groups such ascyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,cyclooctyl, adamantyl, and norbornyl. The term “lower alkyl” refers toan alkyl group of 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms.Exemplary substituents on substituted alkyl groups include hydroxyl,cyano, alkoxy, ═O, ═S, —NO₂, halogen, haloalkyl, heteroalkyl,carboxyalkyl, amine, amide, thioether and —SH.

“Alkoxy” refers to an “—Oalkyl” group, where alkyl is as defined above.A “lower alkoxy” group it tends an alkoxy group containing one to six,more preferably one to four, carbon atoms.

“Alkenyl” refers to a branched, unbranched or cyclic hydrocarbon groupof 2 to 24 carbon atoms containing at least one carbon-carbon doublebond optionally substituted at one or more positions. Examples ofalkenyl groups include ethenyl, 1-propenyl, 2-propenyl (allyl),1-methylvinyl, cyclopropenyl, 1-butenyl, 2-batenyl, isobutenyl,1,4-butadienyl, cyclobutenyl, 1-methylbut-2-enyl, 2-methylbut-2-en-4-yl,prenyl, pent-1-enyl, pent-3-enyl, 1,1-dimethylallyl, cyclopentenyl,hex-2-enyl, 1-methyl-1-ethylallyl, cyclohexenyl, heptenyl,cycloheptenyl, octenyl, cyclooctenyl, decenyl, tetradecenyl,hexadecenyl, eicosenyl, tetracosenyl and the like. Preferred alkenylgroups herein contain 2 to 12 carbon atoms. The term “lower alkenyl”intends an alkenyl group of 2 to 6 carbon atoms, preferably 2 to 4carbon atoms. The term “cycloalkenyl” intends a cyclic alkenyl group of3 to 8, preferably 5 or 6, carbon atoms. Exemplary substituents onsubstituted alkenyl groups include hydroxyl, cyano, alkoxy, ═O, ═S,—NO₂, halogen, haloalkyl, heteroalkyl, amine, thioether and —SH.

“Alkenyloxy” refers to an “-Oalkenyi” group, wherein alkenyl is asdefined above.

“Alkylaryl” refers to an alkyl group that is covalently joined to anaryl group. Preferably, the alkyl is a lower alkyl. Exemplary alkylarylgroups include benzyl, phenethyl, phenopropyl, 1-benzylethyl,phenobutyl, 2-benzylpropyl and the like.

“Alkylaryloxy” refers to an “—Oalkylaryl” group, where alkylaryl is asdefined above.

“Alkenyl” refers to a branched or unbranched hydrocarbon group of 2 to24 carbon atoms containing at least one —C/C— triple bond, optionallysubstituted at one or more positions. Examples of alkynyl groups includeethynyl, n-propynyl, isopropynyl, propargyl, but-2-ynyl,3-methylbut-1-ynyl, octynyl, decynyl and the like. Preferred alkynylgroups herein contain 2 to 12 carbon atoms. The term “lower alkynyl”intends an alkynyl group of 2 to 6, preferably 2 to 4, carbon atoms, andone —C═C— triple bond. Exemplary substituents on substituted alkynylgroups include hydroxyl, cyano, alkoxy, ═O, ═S, —NO₂, halogen,haloalkyl, heteroalkyl, amine, thioether and —SH.

“Antibody” as referenced herein is used in the broadest sense, andspecifically covers monoclonal antibodies (including full lengthmonoclonal antibodies), polyclonal antibodies, multispecific antibodies(e.g., bispecific antibodies), and antibody fragments (e.g., Fab,F(ab′)₂ and Fv) so long as they exhibit binding activity or affinity fora selected antigen.

“Antigen” as used herein refers to any substance capable of eliciting animmune response.

“Amide” refers to —C(O)NR′R″, where R′ and R″ are independently selectedfrom hydrogen, alkyl, aryl, and alkylaryl.

“Amine” refers to an —N(R)R″ group, where R′ and R″ are independentlyselected from hydrogen, alkyl, aryl, and alkylaryl.

“Aryl” refers to an aromatic group that has at least one ring having aconjugated pi electron system and includes carbocyclic, heterocyclic,bridged and/or polycyclic aryl groups, and can be optionally substitutedat one or more positions. Typical aryl groups contain 1 to 5 aromaticrings, which may be fused and/or linked. Exemplary aryl groups includephenyl, furanyl, azolyl, thiofuranyl, pyridyl, pyrimidyl, pyrazinyl,triazinyl, biphenyl, indenyl, benzofuranyl, indolyl, naphthyl,quinolinyl, isoquinolinyl, quinazolinyl, pyridopyridinyl,pyrrolopyridinyl, purinyl, tetraanyi and the like. Exemplarysubstituents on optionally substituted aryl groups include alkyl,alkoxy, alkylcarboxy, alkenyl, alkenyloxy, alkenylcarboxy, aryl,aryloxy, alkylaryl, alkylaryloxy, fused saturated or unsaturatedoptionally substituted rings, halogen, haloalkyl, heteroalkyl, —S(O)R,sulfonyl, —SO₃R, —SR, —NO₂, —NRR′, —OH, —CN, —C(O)R, —OC(O)R, —NHC(O)R.,—(CH₂)_(n)CO₂R or —(CH₂)_(n)CONRR′ where n is 0-4, and wherein R and R′are independently H, alkyl, aryl or alkylaryl.

“Aryloxy” refers to an “—Oaryl” group, where aryl is as defined above.

“Carbocyclic” refers to an optionally substituted compound containing atleast one ring and wherein all ring atoms are carbon, and can besaturated or unsaturated.

“Carbocyclic aryl” refers to an optionally substituted aryl groupwherein the ring atoms are carbon.

“Halo” or “halogen” refers to fluoro, chloro, aroma or iodo. “Halide”refers to the anionic form of the halogens.

“Haloalkyl” refers to an alkyl group substituted at one or morepositions with a halogen, and includes alkyl groups substituted withonly one type of halogen atom as well as alkyl groups substituted with amixture of different types of halogen atoms. Exemplary haloalkyl groupsinclude trihalomethyl groups, for example trifluoromemyl.

“Heteroalkyl” refers to an alkyl group wherein one or more carbon atomsand associated hydrogen atoms) are replaced by an optionally substitutedheteroatom, and includes alkyl groups substituted with only one type ofheteroatom as well as alkyl groups substituted with a mixture ofdifferent types of heteroatoms. Heteroatoms include oxygen, sulfur, andnitrogen. As used herein, nitrogen heteroatoms and sulfur heteroatomsinclude any oxidized form of nitrogen and sulfur, and any form ofnitrogen having four covalent bonds including protonated forms. Anoptionally substituted heteroatom refers to replacement of one or morehydrogens attached to a nitrogen atom with alkyl, aryl, alkylaryl orhydroxyl.

“Heterocyclic” refers to a compound containing at least one saturated orunsaturated ring having at least one heteroatom and optionallysubstituted at one or more positions. Typical heterocyclic groupscontain 1 to 5 rings, which may be fused and/or linked, where the ringseach contain five or six atoms. Examples of heterocyclic groups includepiperidinyl, morpholinyl and pyrrolidinyl. Exemplary substituents foroptionally substituted heterocyclic groups are as for alkyl and aryl atring carbons and as for heteroalkyl at heteroatoms.

“Heterocyclic aryl” refers to an aryl group having at least 1 heteroatomin at least one aromatic ring. Exemplary heterocyclic aryl groupsinclude furanyl, thienyl, pyridyl, pyridazinyl, pyrrolyl, N-loweralkyl-pyrrolo, pyrimidyl, pyrazinyl, triazinyl, tetrazinyl, triazolyl,tetrazolyl, imidazolyl, bipyridyl, tripyridyl, tetrapyridyl, phenazinyl,phenanthrolinyl, purinyl and the like.

“Hydrocarbyl” refers to hydrocarbyl substituents containing 1 to about20 carbon atoms, including branched, unbranched and cyclic species aswell as saturated and unsaturated species, for example alkyl groups,alkylidenyl groups, alkenyl groups, alkylaryl groups, aryl groups, andthe like. The term “lower hydrocarbyl” intends a hydrocarbyl group ofone to six carbon atoms, preferably one to four carbon atoms.

A “substituent” refers to a group that replaces one or more hydrogensattached to a carbon or nitrogen. Exemplary substituents include alkyl,alkylidenyl, alkylcarboxy, alkoxy, alkenyl, alkenylcarboxy, alkenyloxy,aryl, aryloxy, alkylaryl, alkylaryloxy, —OH, amide, carboxamide,carboxy, sulfonyl, ═O, ═S, —-NO₂, halogen, haloalkyl, fused saturated orunsaturated optionally substituted rings, ═S(O)R, —SO₃R, —SR, —NRR′,—OH, —CN, —C(O)R, —OC(O)R, —NHC(O)R, —(CH2)_(n)CO₂R or —(CH2)_(n)CONRR′where n is 0-4, and wherein R and R′ are independently H, alkyl, aryl oralkylaryl. Substituents also include replacement of a carbon atom andone or mare associated hydrogen atoms with an optionally substitutedheteroatom.

“Sulfonyl” refers to —S(O)₂R, where R is alkyl, aryl, —C(CN)═C-aryl,—CH₂CN, alkylaryl, or amine.

“Thioamide” refers to —C(S)NR′R″, where R′ and R″ are independentlyselected from hydrogen, alkyl, aryl, and alkylaryl.

“Thioether” refers to —SR, where R is alkyl, aryl, or alkylaryl.

As used herein, the term “binding pair” refers to first and secondmolecules that bind specifically to each other with greater affinitythan to other components in the sample. The binding between the membersof the binding; pair is typically noncovalent. Exemplary binding pairsinclude immunological binding pairs (e.g., any haptenic or antigeniccompound in combination with a corresponding antibody or binding portionor fragment thereof, for example digoxigenin and anti-digoxigenin,fluorescein and anti-fluorescein, dinitrophenol and anti-dinitrophenol,bromodeoxyuridine and anti-bromodeoxyuridine, mouse immunoglobulin andgoat anti-mouse immunoglobulin) and nonimmunological binding pairs(e.g., biotin-avidin, biotin-streptavidin, hormone [e.g., thyroxine, andcortisol]-hormone binding protein, receptor-receptor agonist orantagonist (e.g., acetylcholine receptor-acetylcholine, or an analogthereof) IgG-protein A, lectin-carbohydrate, enzyme-enzyme cofactor,enzyme-enzyme-inhibitor, and complementary polynucleotide pairs capableof forming nucleic acid duplexes) and the like. One or both member ofthe binding pair can be conjugated to additional molecules.

The terms “polynucleotide,” “oligonucleotide,” “nucleic acid” and“nucleic acid molecule” are used interchangeably herein to refer to apolymeric form of nucleotides of any length, and may compriseribonucleotides, deoxyribonucleotides, analogs thereof, or mixturesthereof. These terms refer only to the primary structure of themolecule. Thus, the terms includes triple-, double- and single-strandeddeoxyribonucleic acid (“DNA”), as well as triple-, double- andsingle-stranded ribonucleic acid (“RNA”). It also includes modified, forexample by alkylation, and/or by capping, and unmodified forms of thepolynucleotide. Additional details for these terms as well as fordetails of base pair formation can be found in U.S. application Ser. No.11/344,942, filed Jan. 31, 2006 which is incorporate herein by referencein its entirety.

“Complementary” or “substantially complementary” refers to the abilityto hybridize or base pair between nucleotides or nucleic acids, such as,for instance, between a sensor peptide nucleic acid and a targetpolynucleotide. Complementary nucleotides are, generally, A and T (or Aand U), or C and G. Two single-stranded polynucleotides or PNAs are saidto be substantially complementary when the bases of one strand,optimally aligned and compared and with appropriate insertions ordeletions, pair with at least about 80% of the bases of the otherstrand, usually at least about 90% to 95%, and more preferably fromabout 98 to 100%.

Alternatively, substantial complementarity exists when a polynucleotideor PNA will hybridize under selective hybridization conditions to itscomplement. Typically, selective hybridization will occur when there isat least about 65% complementary over a stretch of at least 14 to 25bases, preferably at least about 75%, more preferably at least about 90%complementary. See, M. Kanehisa Nucleic Acids Res. 12:203(1984).

“Preferential binding” or “preferential hybridization” refers to theincreased propensity of one polynucleotide or PNA to bind to itscomplement in a sample as compared to a noncomplementary polymer in thesample.

Hybridization conditions for polynucleotides will typically include saltconcentrations of less than about 1M, more usually less than about 500mM and preferably less than about 200 mM. In the case of hybridizationbetween a peptide nucleic acid and a polynucleotide, the hybridizationcan be done in solutions containing little or no salt. Hybridizationtemperatures can be as low as 5° C., but are typically greater than 22°C., more typically greater than about 30° C., and preferably in excessof about 37° C. Longer fragments may require higher hybridizationtemperatures for specific hybridization. Other factors may affect thestringency of hybridization, including base composition and length ofthe complementary strands, presence of organic solvents and extent ofbase mismatching, and the combination of parameters used is moreimportant than the absolute measure of any one alone. Otherhybridization conditions which may be controlled include buffer type,and concentration, solution pH, presence and concentration of blockingreagents to decrease background binding such as repeat sequences orblocking protein solutions, detergent type(s) and concentrations,molecules such as polymers which increase the relative concentration ofthe polynucleotides, metal ion(s) and their concentration(s),chelator(s) and their concentrations, and other conditions known in theart.

“Multiplexing” herein refers to an assay or other analytical methodwhich multiple analytes can be assayed simultaneously.

“Having” is an open ended phrase like “comprising” and “including,” andincludes circumstances where additional elements are included andcircumstances where they are not.

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances where the event or circumstance occurs and instances in whichit does not.

The invention disclosed herein relates generally to assays and complexesincluding multichromophores, and signaling chromophores useful for theidentification of target biomolecules or biomolecules associated withtarget molecules through enhanced signal amplifications.

In general, in one aspect the invention includes multichromophore energytransfer to a dye on a sensor which can be a biomolecule including abioconjugate (e.g., an antibody).

In one embodiment an approach modifying a format as followed in relationto nucleic acid sensor assays as described in Gaylord, Heeger, andBazaar, J. Am. Chem. Soc., 2003 can be followed. Specifically, signalamplification of multichromophore can be based on nonspecificelecostatic binding events to indicate a hybridization event. Anyestablished multichromophore can be chosen as the donor, and one or moredye, preferably a dye with a history of efficient energy transfer, forexample, fluorescein and Cy3, can be chosen as the acceptors. It isenvisioned that the dye can be directly conjugated to a sensor molecule.As shown schematically in FIG. 1, the sensor can be a biomolecule (e.g.,an antibody) in a solution or on a substrate, to which multichromophorecan be added. In the embodiment shown in FIG. 1, a dye can be covalentlylinked (bioconjugated) to an antibody (Y-shaped structure), whichpossesses a net negative charge. Addition of cationic multichromophore(shown as wavy lines) can result in electrostatic binding between themultichromophore and the antibody, bringing the multichromophore and dyeinto close proximity. Distance requirements for fluorescence resonanceenergy transfer (FRET) can thus be met, and excitation of the polymerwith light (shown as hv) results in amplified dye emission. It isenvisioned that the multichromophore can be excited at a wavelengthwhere the dye does not have significant absorbance. In one embodimentthe dye emission can be at a longer wavelength than theinultichromophore emission. In use it is envisioned that an assay methodcan include the steps of providing a sample that is suspected ofcontaining a target biomolecule, providing a sensor conjugated to asignaling chromophore and capable of interacting with the targetbiomolecule, providing a multichromophore that electrostaticallyinteracts with the sensor and upon excitation is capable of transferringenergy to the sensor signaling chromophore and contacting the samplewith the sensor and the multichromophore in a solution under conditionsin which the sensor can bind to the target biomolecule if present. Next,the method can include applying a light source to the sample that canexcite the multichromophore, and detecting whether light is emitted fromthe signaling chromophore.

An example of data produced from the embodiment shown in FIG. 1 ispresented in FIG. 2. As shown in the graph a FITC-labeledmouse-anti-human CD22 antibody can be excited both directly (lower line,labeled FITC) or indirectly through excitation of and electrostaticallybound multichromophore (structure shown in FIG. 2, right) and subsequentenergy transfer via FRET (upper line, labeled Signal amplified bypolymer). The particulars of the experiment included direct excitationof a FITC-labeled mouse-anti-human CD22 antibody (lower line, labeledFITC, 496 nm excitation, [FITC-labeled mouse-anti-human CD22]=1 ng/mL)and multichromophore-amplified dye emission (upper line, 380 nmexcitation, [multichromophore]=1×10⁻⁶ M in repeat units, RU) in 2 mL of1× SSPE. The structure of the donor multichromophore is illustrated tothe right of the graph. Advantageously, energy transfer in the presenceof multichromophore resulted in 5-fold amplification of the dye signalintensity, as compared with direct excitation.

FIG. 3 illustrates a second example of data produced from the embodimentshown in FIG. 1. Here, the graph shows a comparison of optical reportingsignals for direct (lower line, labeled Cy3, 540 nm excitation) andindirect (upper line, 380 nm excitation) excitation of a Cy3-labeleddonkey-anti-mouse secondary antibody. Experimental conditions weresimilar to those for the prior experiment, but with half the volume. Thedonor multichromophore structure is shown in FIG. 3, right side.Multichromophore-amplified dye intensities were 10-fold more intensewhen compared with direct excitation of the dye.

As disclosed herein, electrostatic binding between chargedmultichromophores and dye-labeled antibodies can be a viable approachfor increasing detection sensitivities, for example of a biomoleculetarget. In a further embodiment, covalently attaching themultichromophore to a dyelbiomolecule (e.g., an antibody complex offersseveral advantages including reduced background and improved energytransfer. In the case of direct linkage to a biomolecule, biorecognitionevents, rather than electrostatic binding events, should governmultichromophore presence. In this manner, nonspecific binding ofmultichromophore to biomolecules can be eliminated, reducing anybackground emission resulting from the multichromophore itself. Theahovementioned biomolecules include but are not limited to proteins,peptides, affinity ligands, antibodies, antibody fragments, sugars,lipids, and nucleic acids (as hybridization probes and/or aptamers).

In the case of direct linkage to a dye or biomolecule/dye complex,donor-acceptor distances can be fixed, rather than dependent on thestrength of electrostatic binding, and energy transfer efficiency can besignificantly increased. This has significant consequences in thecontext of improving dye signaling and reducing background fluorescenceassociated with donor-acceptor cross-talk. Cross-talk in this caserefers to the overlap between multichromophore (donor) and dye(acceptor) emission peaks. Multichromophores which bind non-specificallyat distances too great for energy transfer can contribute to thebackground fluorescence (or crosstalk). Shorter (fixed) distancesbetween the donor and acceptor can not only facilitate direct dyeamplification, but also can greatly quench the donor emission. Thisresults in less donor emission at the acceptor emission wavelengths,which subsequently reduces or even eliminates the need for cross-talkcorrection.

In general, in another aspect the invention includes the bioconjugationof multichromophore to affinity ligands (affinity ligands describing abiomolecule that has an affinity for another biomolecule). FIG. 4illustrates a class of materials in which a multichromophore (shown as awavy line) is linked to a dye, biomolecule, or biamolecule/dye complex(labeled X). Linking to the multichromophore can be via a firstfunctionality linker A on the multichromophore that serves as abioconjugation site capable of covalently linking with a secondfunctionality linker A′ linked to a biomolecule and/or dye (see X). Thisarrangement can fix the distance between the multichromophore and X,thereby ensuring only specific interactions between muitichromophore andX. It is envisioned that a biomolecule component X in this embodimentcan be any of the various biomolecules disclosed herein, including butnot limited to an antibody, protein, affinity ligand, or nucleic acid.

It is envisioned that the X in this context can be, but is not limitedto, a dye, fluorescence protein, nanomaterial e.g., Quantum Dot), aconjugate between dye and chemluminescence-generating molecule, aconjugate between fluorescence protein and chemluminescence-generatingmolecule, a conjugate between nanomaterial (e.g., Quantum Dot) andchemluminescence-generating molecule, streptavidin, avidin, enzyme,substrate for an enzyme, substrate analog for an enzyme, receptor,ligand for a receptor, ligand analog for a receptor, DNA, RNA, modifiednucleic acid, DNA aptamer, RNA aptamer, modified nucleic aptamer,peptide aptamer, antibody, antigen, phage, bacterium or conjugate of anytwo of the items described above.

The linking chemistry for A-A′ and B-B′ can include, but not limited to,maleimide/thiol, succimidylester (NHS ester)/amine, azide chemistry,carboxy/EDC (1-Ethyl-3-[3-dimethylaminopropyl]carbodiimideHydrochloride)/amine, amine/Sulfo-SMCC (Sulfosuccinimidyl4-[N-maleimidomethyl]cyclohexane-1-carboxylate)/thiol, and amine/BMPH(N-[β-Maleimidopropionic acid]hydrazide.TFA)/thiol.

In another aspect, the invention includes labeled multichromophores.FIG. 5 shows two examples of labeled multichromophores. In oneembodiment, on the left, a multichromophore (shown as a wavy line) isshown conjugated to an antibody which can be, for example, a 1° or 2°antibody. The conjugate of the multichromophore and the antibody can beused as a reporter, for example, in a assay. Excitation of themultichromophore with light (not shown) can result in multichromophoreemission, indicating the presence of the antibody (1° or 2°). In anotherembodiment shown in FIG. 5 on the right, the multichromophore is labeledwith a dye, for example, a chromophore. In this case, themultichromophore can act as a donor and the dye can act as an acceptorin a FRET process as shown. Here, the multichromophore can act as alight harvester, and excitation of the multichromophore is followed bythe channeling of the excitations to the dye via a FRET process. Thisresults in amplified dye emission (as compared to direct excitation ofthe dye). The fluorescence of the donor multichromophore, in oneembodiment, can be quenched (e.g., >90% quenching).

In general, in another aspect the invention includes a method ofassaying for a target biomolecule or a tagged target biomolecule. Asshown in FIG. 6 in one embodiment a multichromophore (shown as a wavyline) can be linked to a first bioconjugate (shown as a Y-shapedobject), for example, a 2° antibody that is specific for second adye-labeled bioconjugate, for example, a 1° antibody. Here, therecognition event between the 1° and 2° antibody will result in thereduction of distance between the donor multichromophore and acceptordye. After this recognition event, excitation of the donormultichromophore with light (shown as hv) will result in FRET to theacceptor dye (shown as curved arrow), and amplified dye emission (incomparison with direct excitation of the dye) will be observed. In useit is envisioned that an assay method could include providing a samplethat is suspected of containing a target biomolecule by the steps ofproviding a first bioconjugate, for example, a 1° antibody conjugated toa signaling chromophore and capable of interacting with the targetbiomolecule. This is followed by providine a second bioconjugate, forexample, a 2° antibody, conjugated to a multichromophore, wherein thesecond bioconjugate can bind to the first bioconjugate and wherein uponsuch binding excitation of the multichromophore is capable oftransferring energy to the signaling chromophore. Next, the methodincludes contacting the sample with the first bioconjugate in a solutionunder conditions in which the first bioconjugate can bind to the targetbiomolecule if present and contacting the solution with the secondbioconjugate. The method then includes applying a light source to thetarget biomolecule or tagged target biomolecule wherein the light sourcecan excite the multichromophore and subsequently detecting whether lightis emitted from the signaling chromophore.

In general in another aspect, the invention includes a method ofassaying a sample using a multichromophore and a sensor biomoleculecomplex. As shown in FIG. 7, left side, a multichromophore (shown as awavy line) can be conjugated to a first bioconjugate, for example,streptavidin (SA) which has a strong affinity for biotin, In FIG. 7 onthe left, a sensor biomolecule (e.g., an antibody which can be a 1° or2° antibody), is conjugated to both a dye and a second bioconjugate abiotin moiety). After a biorecognition event between the first andsecond bioconjugates (e.g, between SA and biotin), the multichromophoreand dye will be brought into close proximity, and excitation of thedonor multichromophore will result in FRET to the acceptor dye. Dyeemission will indicate the presence of the first bioconjugate (e.g., theantibody). In comparison with direct excitation of the dye,amplification of the dye signal intensity will be observed when excitedindirectly through FRET.

In another embodiment as shown in FIG. 7, right, a sensor biomolecule,for example, a nucleic acid, is conjugated to both a dye and a firstbioconjugate (e.g., a biotin moiety). After a biorecognition eventbetween a second bioconjugate SA) and the first bioconjugate (e.g.,biotin), the multichromophore and dye will be brought into closeproximity, and excitation of the donor multichromophore will result inFRET to the acceptor dye. In comparison with direct excitation of thedye, amplification of the dye signal intensity will be observed whenexcited indirectly through FRET. Dye emission will indicate the presenceof the sensor biomolecule (e.g., a nucleic acid).

A method of using the embodiment shown in FIG. 7 can include the stepsof providing a sample that is suspected of containing a targetbiomolecule, providing a multichromophore comprising a covalently linkedfirst bioconjugate (e.g., SA), providing a sensor biomolecule complexcomprising a sensor biomolecule capable of interacting with the targetmolecule, a signaling chromophore, and covalently linked secondbioconjugate capable of binding with the first bioconjugate, whereinupon such binding excitation of the multichromophore is capable oftransferring energy to the signaling chromophore. The method can furtherinclude the steps of contacting the sample with the sensor biomoleculecomplex in a solution under conditions in which the sensor biomoleculecan bind to the target biomolecule if present, contacting the solutionwith the multichromophore, applying a light source to the sample thatcan excite the multichromophore, and detecting whether light is emittedfrom the signaling chromophore.

In general in another aspect, the invention provides a biorecognitioncomplex for identifying a biomolecule including a bioconjugate asignaling chromophore and a multichromophore. FIG. 8 shows amultichromophore conjugated directly to a dye-labeled bioconjugate,e.g., an antibody (left). FIG. 8 further shows an alternative embodimentwherein a multichromophore is conjugated to a dye-labeled SA (right). Inthe embodiment illustrated on the left, covalent linkages between thebioconjugate (shown as Y-shaped) and the dye and multichromophore ensurethe close proximity of the donor multichromophore and acceptor dye. Upona biorecognition event between the bioconjugate, for example, anantibody, and its target, for example an antigen, excitation of thedonor multichromophore will result in FRET to the acceptor dye. In onealternative embodiment, illustrated in FIG. 8 on the right, themultichromophore and dye remain in fixed, close proximity. As such, upona binding event, for example, between the SA and a biotin moiety,excitation of the donor multichromophore will result in FRET to theacceptor dye, In either embodiment illustrated in FIG. 8, amplified dyeemission should result from multichromophore excitation.

A non-limiting example of a CP structure is shown in FIG. 9. Thebackbone can consists mainly of fluorene-phenylene repeat units andserves as the donor in the FRET process. The CP is functionalized withR1 and R2 groups. Both can serve to solubitize the CP with hydrophilicgroups, including but not limited to quaternary amines or PEG-typefunctionalities, while R2 can also serve to tune the optical propertiesvia energy level modifications. A third co-monomer phenyl groupfunctionalized with a site A allows for bioconjugation to a dye orbiomolecule. The linker A can be but is not limited to a maleimide,thiol, succimidyl ester (or NHS-ester), amine, azide, biotin,avidin/streptavidin, or some other ligand-receptor that will react withan A′ linker that is available on a biomolecule or dye (see e.g., as inFIG. 4).

A unique monomer that allows for the synthesis of the exemplary polymerof FIG. 9 is shown in FIG. 10. This monomer has two sites for Suzukicouplings (see Liu and Bazan. J. Am. Chem. Soc., 2005; Liu and Bazan,Proc. Natl. Acad. Sci., U.S.A., 2005: Bazan, Liu, US ProvisionalApplication No. 60/666,333, filed Sep. 17, 2000, and importantly, a siteA that allows for bioconjugation. Site A can be a bioconjugation siteself, or a precursor, such as a phthalimicle (a protected amine).

Several examples of suitable monomer syntheses are illustrated in FIGS.11 and 12. FIG. 11 schematically shows the synthesis of a monomer with aphthalimide functionality, which serves as a protected amine. FIG. 12describes the synthesis of a monomer with a maleimide, which can bebioconjugated to thiols.

Examples for the syntheses of two monomeric structures forpolymerization follow. The first is a one-step synthesis for a monomerfunctionalized with a protected amine (in the form of a phthalimide) forbioconjugation to succimidyl esters, and the second is a four-stepsynthesis for a monomer functionalized with a maleimide forbioconjugation to thiols.

N-4′-(3″,5″-dibromophenoxy)butylphthalimide or1-(4′-phthalimidobutoxy)3,5-dibromobenzene. 3,5-dibromophenol (970 mg,3.85 mmol) was recrystallized from hexanes. After removal of solvent,N-(4-bromobutyl)phthalimide (1.38 g, 4.89 mmol), K₂CO₃ (1.88 g, 13.6mmol), 18-crown-6 (53 mg, 0.20 mmol), and acetone (20 mL) were added.This was refluxed for 1 hour, and then poured into 100 mL of water. Theaqueous layer was extracted with dichloromethane (4×30 mL). The organiclayers were combined, washed with water, saturated NaHCO₃, and brine,then dried over MgSO₄ and filtered. Removal of solvent yielded a whitesolid, which was purified by column chromatography (4:1 hexanes:CH₂Cl₂)followed by recrystallization in hexanes to yield colorless needles (650mg, 87%).

N-Methoxycarbonylmaleimide. Maleimide (2.00 g, 20.6 mmol) and N-methylmorpholine (2.08 g, 20.6 mmol) in ethyl acetate were cooled to 0° C.Dropwise addition of methylchloroformate (1.4 mL, 20.7 mmol) producedwhite precipitate. The solution was stirred for 1 hour at 0 ° C., afterwhich the solids were removed by filtration. Concentration of thefiltrate yielded a pink oil, which was purified by column chromatography(eluant 3:1 hexanes:ethyl acetate) to yield pale yellow crystals.

N-(ω-hydroxyhexyl)maleimide. 6-amino-1-hexanol and saturated NaHCO₃ (20mL) were cooled to 0°°C. N-Methoxycarbonylmaleimide was added inportions with stiffing. Solids did not fully dissolve. This was stirredfor 30 minutes at 0 ° C. (most solids dissolved after 20 minutes), thenthe ice bath removed and solution stirred for an additional 30 minutes,at which point the solution was pale pink. This was diluted 3-fold withwater and washed with chloroform (3×40 mL), dried over MgSO₄, filtered,and the solvent removed via rotary evaporation.

1-(6′-Bromohexyloxy)-3,5-dibromobenzene. 3,5-Dibromophenol wasrecrystallized from hexanes. After removal of solvent,1,6-dibromohexane, K₂CO₃, 18-crown-6, and acetone were added. This wasrefluxed for 1 hour, and then poured into 100 mL of water. The aqueouslayer was extracted with dichloromethane (4×30 mL). The organic layerswere combined, washed with water, saturated NaHCO₃, and brine, thendried over MgSO₄ and filtered. Removal of solvent yielded an off-whitesolid, which was purified by column chromatography to yield a whitesolid.

1-(6′-(6″Maleimidohexyloxy)hexyloxy)-3,5-dibrornobenzene.N-(ω-hydroxyhexyl)maleimide, 1-(6′-Bromohexyloxy)-3,5-dibromobenzene,K₂CO₃, 18-crown-6, and acetone will be refluxed for 1 hour, and thenpoured into 100 mL of water. The aqueous layer will be extracted withdichloromethane (4×30 mL). The organic layers will be combined, washedwith water, saturated NaHCO₃, and brine, then dried over MgSO₄ andfiltered. Removal of solvent will yield crude material, which will bepurified by column chromatography to yield purified product.

In general, in another aspect the invention provides a multichromophorecomplex including a multichromophore, sensor biomolecule and a signalingchromophore for identifying a target biomolecule. As depicted in FIG.13, in one embodiment a multichromophore can be bioconjugated to both adye and a biomolecule, for example a biorecognition molecule. Usefulbiomolecules can include but are not imited to antibodies, affinityligands, nucleic acids, proteins, nanoparticles or substrates forenzymes. The benefits of covalently linking a dye in proximity to amultichromophore have been described above. By affixing both an acceptordye and a biorecognition molecule to a multichromophore, the benefitsare two fold, by both fixing donor-acceptor distances, such that anacceptor is guaranteed to he within the vicinity of a donormultichromophore (and vice versa), and also increasing the specificityof multichromophore binding to indicate a biorecognition event. Thesecovalent complexes can be made via the monomer and linking chemistriesdescribed herein.

As shown in FIG. 13, left, in one embodiment a multichromophore (wavyline) can be biconjugated to a dye X via linker functionalities A-A′ anda biomolecule Y via linker functionalities B-B′. In an alternativeembodiment shown in FIG. 13, right, a multichromophore can bebioconjugated to a dye X and a biomolecule Y by a tri-functionalizedlinker via linker functionalities A-A′ B-B′, and C-C. In the embodimentillustrated in FIG. 13, the X can be, but is not limited to, a dye,fluorescence protein, nanomaterial (e.g., Quantum Dot), a conjugatebetween dye and chemluminescence-generating molecule, a conjugatebetween fluorescence protein and chemluminescence-generating molecule,or a conjugate between nanomaterial (e.g., Quantum Dot) andchemluminescence-generating molecule. The Y can be, but is not limitedto, a streptavidin, avidin, enzyme, substrate for an enzyme, substrateanalog for an enzyme, receptor, ligand for a receptor, ligand analog fora receptor, DNA, RNA, modified nucleic acid. DNA aptamer, RNA aptamer,modified nucleic aptamer, peptide aptamer, antibody, antigen, phage,bacterium or conjugate of any two of the items described above.

The linking chemistry for A-A′, B-B′ and C-C′ can include, but is notlimited to, maleimide/thiol, succimidylester (NHS ester)/amine, azidechemistry, carboxy/EDC (1 -Ethyl-3-[3-dimethylaminopropyl]carbodiimideHydrochloride/amine, amine/Sulfo-SMCC (Sulfosuccinimidyl4-[N-maleimidomethyl]cyclohexane-1-carboxylate)/thiol, and amine/BMPH(N-[β-Maleimidopropionic acid]hydrazide.TFA)/thiol. A tri-functionallinker such as the commercially available Sulfo-SBEDSulfosuccinimidyl[2-6-(biotinamido)-2-(p-azidobenzamido)-hexanoamido]-ethyl-1,3′-dithiopropionatecan serve well in the three way linkage among X, Y, andmultichromophore.

In use, the embodiments shown in FIG. 13 can be a muhichromophore,complex for identifying a target biomolecule wherein the complexincludes a multichromophore, a signaling chromophore covalently linkedto the multichromophore, and a sensor biomolecule covalently linked tothe multichromophore. The signaling chromophore, of the complex iscapable of receiving energy from he multichromophore upon excitation ofthe multichromophore and the sensor biomolecule is capable ofinteracting with the target biomolecule. It is envisioned that thebiomolecules can include but are not limited to an antibody, protein,affinity ligand, peptide, or nucleic acid.

In general, in another aspect the invention provides a biorecognitioncomplex for identifying a biomolecule wherein the complex includes abioconjugate, a multichromophore and a signaling chromophore. In oneembodiment shown in FIG. 14, left, a multichromophore is conjugated toboth a bioconjugate, for example, an antibody (1° or 2°) and a dye.Covalent linkage between the donor multichromophore and acceptor dyeensures close proximity. Excitation of the donor multichromophoreresults in FRET to the acceptor dye. Where the bioconjugate is anantibody, if the antibody binds to its target (e,g,, antigen), this willbe indicated by dye emission upon donor multichromophore excitation. Inan alternative embodiment, as shown in FIG. 14, right, amultichromophore can be conjugated to both a SA and a dye. Again,covalent linkage between the donor multichromophore and acceptor dyeensure close proximity, and excitation of the donor multichromophoreresults in FRET to the acceptor dye. The SA complex can be used to labelor detect a biotin-labeled biomolecule such as a biotinylated antibodyor nucleic acid. Multichromophore excitation followed by FRET to the dyelabel will result in greatly enhanced detection signals (i.e., greatersensitivity).

FIG. 16 shows an example of a dual-labeled multichromophore (shown aswavy line), bioconjugated to both a reporter dye and a 2° antibody(Y-shaped structure). In an assay, an unlabeled 1° antibody can bind toa an antigen, for example, a target protein (shown as a black triangle).Addition of the 2° antibody, which is conjugated to a multichromophore,and further conjugated to a dye, can bind specifically to the antibody.Optical excitation of the multichromophore can result in energy transferto the dye, and amplified dye emission, in comparison to directexcitation results.

FIG. 17 shows an example of a sandwich-type complex of one embodiment ofthe invention. Here, the multichromophore complex is composed of amultichromophore (shown as wavy line) that is bioconjugated to both adye and a biomolecule, for example, streptavidin (SA). After anunlabeled 1° antibody binds the target protein, shown as a blacktriangle, a biotin-labeled 2° antibody binds specifically to the 1°antibody. In a separate step, addition of the multichromophore complexwill result in specific binding between the biotin and streptavidin, andexcitation of the multichromophore will result in amplified dyeemission, as compared to direct excitation of the dye. Signals arisingfrom dye emission will indicate the presence of the target protein.

In a further aspect, the invention provides for the multiplexing ofdonor energy transfer to multiple acceptors. By using a multichromophoreas a donor in a FRET system, benefits also include the ability tomultiplex. A single donor can transfer energy to several dyes; thus witha single excitation source, the intensity of multiple dyes can bemonitored. This is useful for applications including but not limited tocell imaging (i.e.immunohistochemistry), where the different types ofcells can be monitored by protein-antibody recognition events.

In one embodiment, two dye-labeled antibodies can be incubated with abiological material, for example, a cultured cell line. Antibodies areable to recognize cells with a target protein expressed on its surfaceand specifically bind only to those proteins. By labeling the twoantibodies with different dyes, it is possible, to monitor for theexpression of two different proteins or different cell typessimultaneously. Typically, this would require two scans or images, onceeach with the correct excitation wavelength. As a final step prior toanalysis, these two images would have to be overlaid. By usingantibodies conjugated to both a dye and a multichromophore, oneexcitation wavelength can be used for boat dyes, and a single image willinclude data sets from each of the two antibodies,

A relevant example of this embodiment is shown in FIG. 15, which showsthe emission spectra for a single donor multichromophore with energytransfer to a fluorescein labeled DNA probe (dotted line), energytransfer to a Texas red-labeled DNA probe (dashed line), and energytransfer to both probes (solid line). Additionally, spectra arising fromdirect excitation of the two dyes are shown as solid lines towards thebottom of FIG. 15. Significant amplification of the dyes is seen in allthree cases. Additionally, intense signals are observed for each dye,regardless of the presence or absence of the other dye, indicating goodpotential for multiplexing. Parallel results with protein diagnosticsare envisioned.

Given the potential for multiplexing analysis, it is envisioned that themultichromophore can be linked to a number of dyes, including, but notlimited to, fluorescein, 6-FAM, rhodamine, Texas Red,tetramethylrhodamine, a carboxyrhodamine, carboxyrhodamine 6G,carboxyrhodol, carboxyrhodamine 110, Cascade Blue, Cascade Yellow,coumarin, Cy2®, Cy3®, Cy3.5®, Cy5®, Cy5.5®, Cy-Chrome, phycoerythrin,PerCP (peridinin chlorophyll-a Protein), PerCP-Cy5.5, JOE(6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein), NED, ROX(5-(and-6)-carboxy-X-rhodamine), HEX, Lucifer Yellow, Marina Blue,Oregon Green 488, Oregon Green 500, Oregon Green 514, Alexa Fluor.™ 350,Alexa Fluor®A430, Alexa Fluor® 488, Alexa Fluor® 532, Alexa Fluor® 546,Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 633, Alexa Fluor® 647,Alexa Fluor® 660, Alexa Fluor® 680, 7-amino-4-methylcoumarin-3-aceticacid, BODIPY.™ FL, BODIPY® FL-Br.sub.2, BODIPY® 530/550, BODIPY®558/568, BODIPY® 564/570, BODIPY® 576/589, BODIPY® 581/591, BODIPY®630/650, BODIPY® 650/665, BODIPY® R6G, BODIPY® TMR, BODIPY® TR,conjugates thereof, and combinations thereof.

It is envisioned that the invention described herein can be used toincrease the sensitivity of any of a number of commercially availabletests including but not limited to the OraQuick Rapid HIV-1/2 AntibodyTest, manufactured by OraSure Technologies, Inc. (Bethlehem , Pa.),which is a FDA-approved HIV diagnostic test for oral fluid samples. Thistest can provide screening results with over 99 percent accuracy in aslittle as 20 minutes.

Multichromophores

Light harvesting multichromophore systems can efficiently transferenergy to nearby luminescent species. Mechanisms for energy transferinclude, for example, resonant energy transfer (Forster (orfluorescence) resonance energy transfer, FRET), quantum charge exchange(Dexter energy transfer) and the like. Typically, however, these energytransfer mechanisms are relatively short range, and close proximity ofthe light harvesting multichromophore system to the signalingchromophore is required for efficient energy transfer. Amplification ofthe emission can occur when the number of individual chromophores in thelight harvesting multichromophore system is large; emission from afluorophore can be more intense when the incident light (the “pumplight”) is at a wavelength which is absorbed by the light harvestingmultichromopliore system and transferred to the fluorophore than whenthe fluorophore is directly excited by the pump light.

The multichromophores used in the present invention can be chargeneutral, cationic or anionic. in some embodiments the multichromoplioresare polycationic multichromophores.

In embodiments wherein the multichromophore is polycationic they caninteract with a biomolecule comprising multiple anionic groups, e.g.polysaccharides, polynucleotides, peptides, proteins, antibodies, etc.In some embodiments, the multichromophore can interact with a targetantibody or polynucleotide electrostatically and thereby bring asignaling chromophore on an uncharged sensor polyirucle,otide intoenergy-receiving proximity by virtue of antibody-antigen recognition orhybridization between a sensor polynucleotide and a targetpolynucleotide. Any polycationic multichromophore that can absorb lightand preferably emit or transfer energy can be used in the methodsdescribed. Exemplary multichromophores that can be used includeconjugated polymers (CP), saturated polymers or dendrimers incorporatingmultiple chromophores in any viable manner, and semiconductornanocrystals (SCNCs). The CP, saturated polymers and dendrimers can beprepared to incorporate multiple cationic species or can be derivatizedto render them polycationic after synthesis; semiconductor nanocrystalscan be rendered polycationic by addition of cationic species to theirsurface. In some embodiments, the polycationic multichromophore is notdetected by its ability to transfer energy when excited, and thusmethods involving such detection schemes do not require themultichromophore to emit or transfer energy.

In some embodiments, the multichromophore is a CP. In a particularembodiment, the CP is one that comprises “low bandgap repeat units” of atype and in an amount that contribute an absorption to the polymer inthe range of about 450 nm to about 1000 nm. The low bandgap repeat unitsmay or may not exhibit such an absorption prior to polymerization, butdoes introduce that absorption when incorporated into the conjugatedpolymer. Such absorption characteristics allow the polymer to be excitedat wavelengths that produce less background fluorescence in a variety ofsettings, including in analyzing biological samples and imaging and/ordetecting molecules. Shifting the absorbance of the CP to a lower energyand longer wavelength thus allows for more sensitive and robust methods.Additionally, many commercially available instruments incorporateimaging components that operate at such wavelengths at least in part toavoid such issues. For example, thermal cyders that perform real-timedetection during amplification reactions and microarray readers areavailable which operate in this region. Providing polymers that absorbin this region allows for the adaptation of detection methods to suchformats, and also allows entirely new methods to be performed.

Incorporation of repeat units that decrease the band gap can produceconjugated polymers with such characteristics. Exemplary optionallysubstituted species which result in polymers that absorb light at suchwavelengths include 2,1,3-benzothiadiazole, benzoselenadiazole,benzotellurodiazole, naphthoselenadiazole,4,7-di(thien-2-yl)-2,1,3-benzothiadiazole, squaraine dyes, quinoxalines,low bandgap commercial dyes, olefins, and cyano-substituted olefins andisomers thereof. Further details relating to the composition, structure,properties and synthesis of suitable multichromophores can be found inU.S. Provisional Application No. 60/642,901. filed Jan. 10, 2005 andU.S. patent application Ser. No. 11/329,495, filed Jan. 10, 2006, nowpublished as US 2006-0183140 A1, which are both incorporated herein byreference.

Multichromophores can be described as a set of covalently boundchromophoric units or a covalent collection of chromophores.Multichromophores can include, but are not limited to, linearstructures, such as, conjugated polymers (CPs) and dendritic structures(Wang, Gaylord, and Bazan, Adv. Mater., 2004, Wang. Hong, and Bazan,Org. Lett., 2005).

FIG. 18 illustrates a general structure for a CP as a linearmultichromophore. In one embodiment such a CP could be comprised ofthose units described in Tables 1 and 2 or Scheme 1 of U.S. patentapplication Ser. No. 10/666,333: Conformational Flexible CationicConjugated Polymers, by Liu and Bazan, and also include monomerscontaining one or more unique bioconjugation sites as depicted in FIG.10 herein. The CP preferably contains at least about 0.01 mol % of thebioconjugation site, and may contain at least about 0.02 mol %, at leastabout 0.05 mol %, at least about 0.1 mol %, at least about 0.2 mol %, atleast about 0.5 mol %, at least about 1 mol %, at least about 2 mol %,at least about 5 mol %, at least about 10 mol %, at least about 20 mol%, or at least about 30 mol %. The CCP may contain up to 100 mol % ofthe bioconjugation site, and may contain about 99 mol % or less, about90 mol % or less, about 80 mol % or less, about 70 mol % or less, about60 mol % or less, about 50 mol % or less, or about 40 mol % or less.

In FIG. 18, the units CP1, CP2, CP3, and CP4 are optionally substitutedconjugated polymer segments or oligorneric structures, and may be thesame or different from one another. CP1, CP2, CP3, and CP4 may bearomatic repeat units, and may be selected from the group consisting ofbenzene, naphthalene, anthracene, fluorene, thiophene, furan, pyridine,and oxadiazole, each optionally substituted. Additionally, CP3 and CP4can contain one or more unique bioconjugation sites, linked by a linkerL as in FIG. 3h . These bioconjugation sites can be, but are nor limitedto, maleimide, thiol, succimidylester (NHS ester), amine, azidechemistry, carboxy/EDC (1-Ethyl-3-[3-dimethylaminopropyl]carbodiimideHydrochloride, Sulfo-SMCC (Sulfosuccinimidyl4-[N-maleimidomethyl]cyclohexane-1-carboxylate), amine/BMPH(N-[β-Maleimidopropionic acid]hydrazide.TFA), or Sulfo-SPEDSulfosuccinimidyl[2-6-(biotinamido)-2-(p-azidobenzamido)-hexanoamido]-ethyl-1,3′-dithiopropionate,which can serve as a three way linkage among X, Y, and CP in FIG. 13.

Typical aromatic repeat units are shown in Table 1, and representativepolymeric segments and oligo structures are shown in Table 2 of U.S.patent application Ser. No. 10/666,333: Conformationally FlexibleCationic Conjugated Polymers by Liu and Bazan.

FIG. 18 contains CP3 and CP4, which can be angled linkers (metafashion), and can be mono- or polycyclic optionally substituted arylgroups having 5 to 20 atoms. The CP3 and CP4 units may be evenly orrandomly distributed along the polymer main chain.

CP1, CP2, C3 and CP4 are each optionally substituted at one or morepositions with one, or more groups selected from —R1-A, R2-B, R3-C and—R4-D, which may be attached through bridging functional groups -E- and—F—, with the proviso that the polymer as a whole must be substitutedwith a plurality of cationic, anionic, or charge neutral water-solublegroups.

R1, R2, R3 and R4 are independently selected from alkyl, alkenyl,alkoxy, alkynyl, and aryl, alkylaryl, arylalkyl, and polyalkylene oxide,each optionally substituted, which may contain one or more heteroatoms,or may be not present. R1, R2, R3 and R4 can be independently selectedfrom C1-22 alkyl, C1-22 alkoxy, C1-22 ester, polyalkylene oxide havingfrom 1 to about 22 carbon atoms, cyclic crown ether having from Itoabout 22 carbon atoms, or not present. Preferably, R1, R2, R3 and R4 maybe selected from straight or branched alkyl groups having 1 to about 12carbon atoms, or alkoxy groups with 1 to about 12 carbon atoms. It is tobe understood that more than one functional group may be appended to therings as indicated in the formulas at one or more positions.

A, B, C and D are independently selected from H, —SiR′R″R′″, —N+R′R″R′″,a guanidinium group, histidine, polyamine, a pyridinium group, and asulfonium group. R′, R″ and R′″ are independently selected from thegroup consisting of hydrogen, C₁₋₁₂ alkyl and C₁₋₁₂ alkoxy and C₃₋₁₂cycloalkyl. It is preferred that R′, R″ and R″ are lower alkyl or loweralkoxy groups.

E and F are independently selected from not present, —O—, —S—, —C(O)—,—C(O)O—, —C(R)(R′)—, —N(R′)—, and —Si(R′)(R″), wherein R′ and R″ are asdefined above.

X is O, S, Se, —N(R′)— or —C(R′)(R″)—, and Y and Z are independentlyselected from —C(R)═ and —N═, where R, R′ and R″ are as defined above.

FIG. 19 shows a CP composed of a backbone containing fluorene units andaromatic units 1, 2, and 3. The units 1, and 2 may be, but are notlimited to, the structures shown in FIG. 20. Unit 3 contains abioconjugation site. The R1 functionality is noted as a solubilizinggroup, and can be, but is not limited to, charged alkyl functionalities(i.e., (CH2)nNMe3Br, or (CH2)nSO3Na) or hydrophilic groups ethyleneglycol units, (OCH2CH2)n).

The π-conjugated units 1, 2, and 3 from FIG. 19 include those describedin Tables 1 and 2 and Scheme 1 of Liu and Bazan, U.S. patent applicationSer. No. 10/666,333: Conformationally flexible Cationic ConjugatedPolymers. FIG. 20 shows several specific examples of π-conjugated unitsthat may be contained within a general CP structure, depicted in FIG.19, with asterisks depicting points of covalent binding to the CPbackbone. These units include benzene units connected to the CP backbonein a typical para fashion (a, b, and e) or connected in a meta fashion,which allows for more flexibility within the CP backbone (c and d).These units can be functionalized with moieties that alter theelectronic structure (b, d, and e), including donating groups (alkoxy orethylene glycol units) and withdrawing groups (fluorine) or improvewater solubility (e) with hydrophilic ethylene glycol units or chargedgroups, such as quaternary amines or sulfonates. Also included are unitssuch as thiophenes (f) and benzothiadiazole groups (g), which serve as ameans to alter electronic structure. These units can be also befunctionalzed as described above. The unit h is contains a specificbioconjugation site A, for example, maleimide, which is covalently boundto the π-conjugated segment via a linker L, for example, an alkoxygroup, and may be incorporated into the backbone of a CP in a ortho,para, or meta fashion.

Several variations of specific polymeric structures include those shownin FIG. 21, which contain a percentage of units with a maleimicle arsuccimidyl ester bioconjugation site linked via ether and alkoxylinkages.

Conjugated polymers useful in the present invention include but are notlimited to the following:

Antigen-Antibody Interactions

The interactions between antigens and antibodies are the same as forother non-covalent protein-protein interactions. In general, four typesof binding interactions exist between antigens and antibodies: (i)hydrogen bonds, (ii) dispersion forces, (iii) electrostatic forcesbetween Lewis acids and Lewis bases, and (iv) hydrophobic interactions.Certain physical forces contribute to antigen-antibody binding, forexample, the fit or complimentary of epitope shapes with differentantibody binding sites. Moreover, other materials and antigens maycross-react with an antibody, thereby competing for available freeantibody.

Measurement of the affinity constant and specificity of bindingbetweenantigen and antibody is a pivotal element in determining theefficacy of an immunoassay, not only for assessing the best antigen andantibody preparations to use but also for maintaining quality controlonce the basic immunoassay design is in place.

Antibodies

Antibody molecules belong to a family of plasma proteins calledimmunoglobulins, whose basic building block, the immunoglobulin fold ordomain, is used in various forms in many molecules of the immune systemand other biological recognition systems. A typical immunoglobulin hasfour polypeptide chains, containing an antigen binding region known as avariable region and a non-varying region known as the constant region.

Native antibodies and immunoglobulins are usually heterotetramericglycoproteins of about 150,000 Daltons, composed of two identical light(L) chains and two identical heavy (H) chains. Each light chain islinked to a heavy chain by one covalent disulfide bond, while the numberof disulfide linkages varies between the heavy chains of differentimmunoglobulin isotypes. Each heavy and light chain also has regularlyspaced intrachain disulfide bridges. Each heavy chain has at one end avariable domain (VH) followed by a number of constant domains. Eachlight chain has a variable domain at one end (VL) and a constant domainat its other end. The constant domain of the light chain is aligned withthe first constant domain of the heavy chain, and the light chainvariable domain is aligned with the variable domain of the heavy chain.

Depending on the amino acid sequences of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are at least five (5) major classes of immunoglobulins: IgA, IgD,IgE, IgG and IgM, and several of these may be further divided intosubclasses (isotypes), e.g. IgG-1, IgG-2, IgG-3 and IgG-4; IgA-1 andIgA-2. The subunit structures and three-dimensional configurations ofdifferent classes of immunoglobulins are well known. Further detailsregarding antibody structure, function, use and preparation arediscussed in U.S. Pat. No. 6,998,241, issued Feb. 14, 2006, the entirecontents of which are incorporated herein by reference.

Sandwich Assays

Antibody or multiple antibody sandwich assays are well known to thoseskilled in the art including a disclosed in U.S. Pat. No. 4,486,530,issued Dec. 4, 1984, and references noted therein. The structuresdescribed in FIGS. 6, 7, 8 and 14 can be used directly as described orin various sandwich configurations. A sandwich configuration or asandwich assay refers to the use of successive recognition events tobuild up layers of various biomolecules and reporting elements to signalthe presence of a particular biomolecule, for example a targetbiomolecule or a target-associated biomolecule. A standard example ofthis would be the successive use of antibodies. In these assays, aprimary antibody binds the target, the secondary antibody binds theprimary, a third antibody can bind the secondary and so on. With eachsuccessive layer additional reporting groups can be added. Anotherstrategy is using a repetitive addition of alternating layers of two (ormore) mutually-recognizable components, or more than two components in achain-recognition relationship, which comprise one or both of thecomponents in a form of multimeric structure. In such a setup, one ormore of the functional group(s) in each of the multimeric structure canbe labeled with reporting group(s) and the unoccupied functionalgroup(s) can serve as the recognition site for the other component(s),and this system will subsequently provide a platform for signalamplification. A typical example of this approach is the use ofstreptavidin-reporter conjugate and biotinylated anti-streptavidinantibody. In such assays, a biotinylated sensor molecule (nucleic acidor antibody) can be used to bind a target biomolecule, which issubsequently recognized by a detection system containing astreptavidin-reporter conjugate and biotinylated anti-streptavidinantibody. The sandwich structure in this case can be built up bysuccessive rounds of biotinylated antibodies and labeled streptavidincomplexes interaction to achieve the signal amplification. With anadditional conjugation of a multichromophore to either the biotinylatedantibody or the streptavidin-reporter complex, it is possible to furtherincrease the signal output. In essence, the integration of amultichromophore in this type of signal amplification system can furtheramplify signals to a higher level.

The bioconjugated polymer complexes described in FIGS. 6, 7, 8, 14, 16and 17 can be used to create optically enhanced sandwich assays bydirectly integrating a light harvesting multichromophore into commonlyutilized recognition elements. The benefits of the multichromophoreconjugated structures can also be applied directly to the primary targetrecognition elements without the need for successive recognitionelements. For example, a primary antibody can be directly conjugated tomultichromophore-dye complex such as shown in FIG. 14. Such a complexcan be used to directly probe the presence of a target hiomolecule.

Polynucleotides

Amplified target polynucleotides may be subjected to post amplificationtreatments. For example, in some cases, it may be desirable to fragmentthe target polynucleotide prior to hybridization in order to providesegments which are more readily accessible. Fragmentation of the nucleicacids can be carried out by any method producing fragments of a sizeuseful in the assay being performed; suitable physical, chemical andenzymatic methods are known in the art.

An amplification reaction can be performed under conditions which allowthe sensor polynucleotide to hybridize to the amplification productduring at least part of an amplification cycle. When the assay isperformed in this manner, real-time detection of this hybridizationevent can take place by monitoring for light emission duringamplification.

Real time PCR product analysis (and related real timereverse-transcription PCR) provides a well-known technique for real dinePCR monitoring that has been used in a variety of contexts, which can beadapted for use with the methods described herein (see, Laurendeau etal. (1999) “TaqMan PCR-based gene dosage assay for predictive testing inindividuals from a cancer family with INK4 locus haploinsufficiency”Clin Chem 45(7):982-6 Laurendeau et al. (1999) “Quantitation of MYC geneexpression in sporadic breast tumors with a real-time reversetranscription-PCR assay” Clin Chem 59(12):2759-65; and Kreuter et al.(1999) “LightCycler technology for the quantitation of bcr/abl fusiontranscripts” Cancer Research 59(13):3171-4, all of which areincorporated by reference).

The Sample

In principle, the sample can be any material suspected of containing anaggregant capable of causing aggregation of the aggregation sensor. Insome embodiments, the sample can be any source of biological materialwhich comprises polynucleotides that can be obtained from a livingorganism directly or indirectly, including cells, tissue or fluid, andthe deposits left by that organism, including viruses, mycoplasma, andfossils. The sample may comprise an azgregant prepared through syntheticmeans, in whole or in part. Typically, the sample is obtained as ordispersed in a predominantly aqueous medium. Nonlimiting examples of thesample include blood, urine, semen, milk, sputum, mucus, a buccal swab,a vaginal swab, a rectal swab, an aspirate, a needle biopsy, a sectionof tissue obtained for example by surgery or autopsy, plasma, serum,spinal fluid, lymph fluid, the external secretions of the skin,respiratory, intestinal, and genitourinary tracts, tears, saliva,rumors, organs, samples of in vitro cell culture constituents (includingbut not limited to conditioned medium resulting from the growth of cellsin cell culture medium, putatively virally infected cells, recombinantcells, and cell components), and a recombinant library comprisingpolynucleotide sequences.

The sample can be a positive control sample which is known to containthe aggregant or a surrogate therefore. A negative control sample canalso be used which, although not expected to contain the aggregant, issuspected of containing it (via contamination of one or more of thereagents) or another component capable of producing a false positive,and is tested in order to confirm the lack of contamination by thetarget polynucleotide of the reagents used in a given assay, as well asto determine whether a given set of assay conditions produces falsepositives (a positive signal even in the absence of targetpolynucleotide in the sample).

The sample can be diluted, dissolved, suspended, extracted or otherwisetreated to solubilize and/or purify any target polynucleotide present orto render it accessible to reagents which are used in an amplificationscheme or to detection reagents. Where the sample contains cells, thecells can be lysed or permeabilized to release the polynucleotideswithin the cells. One step permeabilization buffers can be used to lysecells which allow further steps to be performed directly after lysis,for example a polymerase chain reaction.

Signaling Chromophores

In some embodiments, a signaling chromophore or fluorophore may beemployed, for example to receive energy transferred from an excitedstate of an optically active unit, or to exchange energy with a labeledprobe, or in multiple energy transfer schemes. Fluorophores useful inthe inventions described herein include any substance which can absorbenergy of an appropriate wavelength and emit or transfer energy. Formultiplexed assays, a plurality of different fluorophores can be usedwith detectably different emission spectra. Typical fluorophores includefluorescent dyes, semiconductor nanocrystals, lanthanide chelates, andgreen fluorescent protein.

Exemplary fluorescent dyes include fluorescein, 6-EAM, rliodamine, TexasRed, tetramethylrhodamine, a carboxyrhodamine, carboxyrhodamine 6G,carboxyrhodol, carboxyrhodamine 110, Cascade Blue, Cascade Yellow,coumarin, Cy2®, Cy3®, Cy3.5®, Cy5®, Cy5.5®, Cy-Chrome, phycoerythrin.PerCP (peridinin chlorophyll-a Protein), PerCP-Cy5.5. JOE(6-carboxy-4′,5′-dichloro-2′,7′-dimelhoxyfluorescein), NED, ROX(5-(and-6)-carboxy-X-rhodamine), HEX, Lucifer Yellow, Marina Blue,Oregon Green 488, Oregon Green 500, Oregon Green 514, Alexa Fluor® 350,Alexa Fluor® 430, Alexa Fluor® 488, Alexa Fluor® 532, Alexa Fluor® 546,Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 633, Alexa Fluor® 647,Alexa Fluor® 660, Alexa Fluor® 680, 7-amino-4-methylcourmarin-3-aceticacid, BODIPY® FL, BODIPY® FL-Br₂, BODIPY® 530/550, BODIPY® 558/568,BODIPY® 564/570, BODIPY® 576/589, BOLYIPY® 581/591, BODIPY® 630/650,BODIPY® 650/665, BODPY® R6G, BODIPY® TMR, BODIPY® TR, conjugatesthereof, and combinations thereof. Exemplary lanthanide chelates includeeuropium chelates, terbium chelates and samarium chelates.

A wide variety of fluorescent semiconductor nanocrystals (“SCNCs”) areknown in the art; methods of producing and utilizing semiconductornanocrystals are described in: PCT Publ. No. WO 99/26299 published May27,1999, inventors Bawendi et al.; U.S. Pat. No. 5,990,479 issued Nov.23, 1999 to Weiss et al.; and Buchez et al., Science 281:2013, 1998.Semiconductor nanocrystals can be obtained with very narrow emissionbands with well-defined peak emission wavelengths, allowing for a largenumber of different SCNCs to be used as signaling chromophores in thesame assay, optionally in combination with other non-SCNC types ofsignaling chromophores.

Exemplary polynucleotide-specific dyes include acridine orange, acridinehomodimer, actinomycin D, 7-aminoactmomycin D (7-AAD),9-amino-6-chlor-2-methoxyacridine (ACMA), BOBO™-1 iodide (462/481),BOBO™-3 iodide (570/602), BO-PRO™-1 iodide (462/481), BO-PRO™-3 iodide(575/599), 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI),4′,6-diamidino-2-phenytindole, dihydroehloride (DAPI),4′,6-diamidino-2-phenylindole, dilactate (DAPI, dilactate),dihydroethidium (hydroethidine), dihydroethidium (hydroethidine),dihydroethidium (hydroethidine), ethidium bromide, ethidium diazidechloride, ethidium homodimer-1 (EthD-1), ethidium homodimer-2 (EthD-2),ethidium monoazide bromide (EMA), hexidium iodide, Hoechst 33258,Hoechst 33342, Hoechst 34580, Hoechst 5769121, hydroxystilbamidine,methanesulfonate, JOJO™-1 iodide (529/545), JO-PRO™-1 iodide (530/546),LOLO™-1 iodide (565/579), LO-PRO™-1 iodide (567/580), NeuroTrace™435/455, NeuroTrace™ 500/525, NeuroTrace™ 515/535, NeuroTrace™ 530/615,NeuroTrace™ 640/660, OliGreen, PicoGreen™ ssDNA, PicoGreen® dsDNA,POPO™-1 iodide (434/456), POPO™-3 iodide (534/570), PO-PRO™-1 iodide(435/455), PO-PRO™-3 iodide (539/567), propidium iodide, RiboGreen®,SlowFade®, SlowFade® Light, SYBR® Green I, SYBR® Green II, SYBR® Gold,SYBR® 101, SYBR® 102, SYBR® 103, SYBR® DX, TO-PRO®-1, TO-PRO®-3,TO-PRO®-5, TOTO®-1, TOTO®-3, YO-PRO®-1 (oxazole yellow), YO-PRO®-3,YOYO®-1, YOYO®-3, TO, SYTOX® Blue, SYTOX® Green, SYTOX® Orange, SYTO® 9,SYTO® BC, SYTO® 40, SYTO® 41, SYTO® 42, SYTO® 43, SYTO® 44, SYTO® 45,SYTO® Blue, SYTO® 11, SYTO® 12, SYTO® 13, SYTO® 14, SYTO® 15, SYTO® 16,SYTO® 20, SYTO® 21, SYTO® 22, SYTO® 23, SYTO® 24, SYTO® 25, SYTO® Green,SYTO® 80, SYTO® 81, SYTO® 82, SYTO® 83. SYTO® 84, SYTO® 85, SYTO®Orange, SYTO® 17, SYTO® 59, SYTO® 60, SYTO® 61, SYTO® 62, SYTO® 63,SYTO® 64, SYTO® Red, netropsin, distamycin, acridine orange,3,4-benzopyrene, thiazole orange, TOMEHE, daunomycin, acridine,pentyl-TOTAB, and butyl-TOTIN. Asymmetric cyanine dyes may be used asthe polynucleotide-specific dye. Other dyes of interest include thosedescribed by Geierstanger, B. H. and Wemmer, D. E., Annu. Rev. Vioshys.Biomol. Struct. 1995,24,463-493, by Larson. C. J. and Verdine, G. L.,Bioorganic Chemistry: Nucleic Acids, Hecht, S. M., Ed., OxfordUniversity Press: New York, 1996; pp 324-346, and by Glumoff, T. andGoldman, A. Nucleic Acids in Chemistry and Biology, 2^(nd) ed.,Blackburn, G. M. and Gait, M. J., bis., Oxford University Press: Oxford,1996, pp375-441. The polynucleotide-specific dye may be an intercalatingdye, and may be specific for double-stranded polynucleotides. Other dyesand fluorophores are described at www.probes.com(Molecular Probes,Inc.).

The term “green fluorescent protein” refers to both native Aequoreagreen fluorescent protein and mutated versions that have been identifiedas exhibiting altered fluorescence characteristics, including alteredexcitation and emission maxima, as well as excitation and emissionspectra of different shapes (Delagrave, S. et al, (1995) Bio/Technology13:151-154; Heim, R. et al. (1994) Proc. Natl. Acad. Sci. USA91:12501-12504; Heim, R. et al, (1995) Nature 373:663-664). Delgrave etal. isolated mutants of cloned Aequorea victoria GFP that hadred-shifted excitation spectra. Bio/Technology 13:151-154 (1995), Heim,R. et al. reported a mutant (Tyr66 to His) having a blue fluorescence(Proc. Natl. Acad. Sci. (1994) USA 91:12501-12504).

The Substrate

In some embodiments, an assay component can be located upon a substrate.The substrate can comprise a wide range of material, either biological,nonbiological, organic, inorganic, or a combination of any of these. Forexample, the substrate may be a polymerized Langmuir Blodgett film,functionalized glass, Si, Ge, GaAs, GaP, SiO₂, SiN₄, modified silicon,or any one of a wide variety of gels or polymers such as(poly)tetrafluoroethylene, (poly)vinylidenedifluoride, polystyrene,cross-linked polystyrene, polyacrylic, polylactic acid, polyglycolicacid, poly(lactide coglycolide), polyanhydrides, poly(methylmethacrylate), polyethylene-co-vinyl acetate), polysiloxanes, polymericsilica, latexes, dextran polymers, epoxies, polycarbonates, orcombinations thereof. Conducting polymers and photoconductive materialscan be used.

Substrates can be planar crystalline substrates such as silica basedsubstrates (e,g, glass, quartz, or the like), or crystalline substratesused in, e.g., the semiconductor and microprocessor industries, such assilicon, gallium arsenide, indium doped GaN and the like, and includessemiconductor nanocrystals.

The substrate can take the form of a photodiode, an optoelectronicsensor such as an optoelectronic semiconductor chip or optoelectronicthin-film semiconductor, or a biochip. The location(s) of probe(s) onthe substrate can be addressable; this can be done in highly denseformats, and the location(s) can be microaddressable or nanoaddressable.

Silica aerogels can also be used as substrates, and can be prepared bymethods known in the art. Aerogel substrates may be used as freestanding; substrates or as a surface coating for another substratematerial.

The substrate can take any form and typically is a plate, slide, bead,pellet, disk, particle, microparticle, nanoparticle, strand,precipitate, optionally porous gel, sheets, tube, sphere, container,capillary, pad, slice, film, chip, multiwell plate or dish, opticalfiber, etc. The substrate can be any form that is rigid or semi-rigid.The substrate may contain raised or depressed regions on which an assaycomponent is located. The surface of the substrate can be etched usingwell known techniques to provide for desired surface features, forexample trenches, v-grooves, mesa structures, or the like.

Surfaces on the substrate can be composed of the same material as thesubstrate or can be made from a different material, and can be coupledto the substrate by chemical or physical means. Such coupled surfacesmay be composed of any of a wide variety of materials, for example,polymers, plastics, resins, polysaccharides, silica or silica-basedmaterials, carbon, metals, inorganic glasses, membranes, or any of theabove-listed substrate materials. The surface can be opticallytransparent and can have surface Si—OH functionalities, such as thosefound on silica surfaces.

The substrate and/or its optional surface can be chosen to provideappropriate characteristics for the synthetic and/or detection methodsused. The subsuate and/or surface can be transparent to allow theexposure of the substrate by light applied from multiple directions. Thesubstrate and/or surface may be provided with reflective “mirror”structures to increase the recovery of light.

The substrate and/or its surface is generally resistant to, or istreated to resist, the conditions to which it is to be exposed in use,and can be optionally treated to remove any resistant material afterexposure to such conditions.

Polynucleotide probes can be fabricated on or attached to the substrateby any suitable method, for example the methods described in U.S. Pat.No. 5,143,854, PCT Publ. No. WO 92/10092, U.S. patent application Ser.No. 7/624,120, filed Dec. 6, 1990 (now abandoned), Fodor et al.,Science, 251: 767-777 (1991), and PCT Publ. No. WO 90/15070), Techniquesfor the synthesis of these arrays using mechanical synthesis strategiesare described in, e.g., PCT Publication No. WO 93/09668 and U.S. Pat.No. 5,384,261.

Still further techniques include bead based techniques such as thosedescribed in PCT Appl. No. PCT/U.S. patent application Ser. No. 93/04145and pin based methods such as those described in U.S. Pat. No.5,288,514.

Additional flow channel or spotting methods applicable to attachment ofsensor polynucleotides to the substrate are described in U.S. patentapplication Ser. No. 7/980,523, filed Nov. 20, 1992, and U.S. Pat. No.5,384,261. Reagents are delivered to the substrate by either (1) flowingwithin a channel defined on predefined regions or (2) “spotting” onpredefined regions. A protective coating such as a hydrophilic orhydrophobic coating (depending upon the nature of the solvent) can beused over portions of the substrate to be protected, sometimes incombination with materials that facilitate wetting by the reactantsolution in other regions. In this manner, the flowing solutions arefurther prevented from passing outside of their designated flow paths.

Typical dispensers include a micropipette optionally roboticallycontrolled, an ink-jet printer, a series of tubes, a manifold, an arrayof pipettes, or the like so that various reagents can be delivered tothe reaction regions sequentially or simultaneously.

The substrate or a region thereof may be encoded so that the identity ofthe sensor located in the substrate or region being queried may bedetermined. Any suitable coding scheme can be used, for example opticalcodes, RFID tags, magnetic codes, physical codes, fluorescent codes, andcombinations of codes.

Excitation and Detection

Any instrument that provides a wavelength that can excite theaggregation sensor and is shorter than the emission wavelength(s) to bedetected can be used for excitation. Commercially available devices canprovide suitable excitation wavelengths as well as suitable detectioncomponents.

Exemplary excitation sources include a broadband UV light source such asa deuterium lamp with an appropriate filter, the output of a white lightsource such as a xenon lamp or a deuterium lamp after passing through amonochromator to extract out the desired wavelengths, a continuous wave(cw) gas laser, a solid state diode laser, or any of the pulsed lasers.Emitted light can be detected through any suitable device or technique;many suitable approaches are known in the art. For example, afluorimeter or spectrophotometer may be used to detect whether the testsample emits light of a wavelength characteristic of the signalingchromophore upon excitation of the multichromophore.

Compositions of Matter

Also provided are compositions of matter of any of the moleculesdescribed herein in any of various forms. The multichromophores andcomplexes including multichromophores as described herein may beprovided in purified and/or isolated form. The multichromophores andcomplexes including multichromophores may be provided in crystallineform.

The multichromophores and complexes including multichromophores may beprovided in solution, which may be a predominantly aqueous solution,which may comprise one or more of the additional solution componentsdescribed herein, including without limitation additional solvents,buffers, biomolecules, polynucleotides, fluorophores, etc. Themultichromophores and complexes including multichromophores can bepresent in solution at a concentration at which a first emission fromthe first optically active units can be detected in the absence ofbiomolecule, target or a biomolecule associated therewith. The solutionmay comprise additional components as described herein, includinglabeled probes such as fluorescently labeled antibodies orpolynucleotides, specific for a species of a class of biomolecule targetor a biomolecule associated therewith for the multichromophores andcomplexes including mutltichromophores.

The muitichromophores and complexes including multichromophores may beprovided in the form of a film. The compositions of matter may beclaimed by any property described herein, including by proposedstructure, by method of synthesis, by absorption and/or emissionspectrum, by elemental analysis, by NMR spectra, or by any otherproperty or characteristic.

In some embodiments expression of a gene is detected in a sample. In afurther embodiment, a measured result of detecting a biomolecule targetor a biomolecule associated therewith can be used to diagnose a diseasestate of a patient. In yet another embodiment the detection method ofthe invention can further include a method of diagnosing a diseasestate. In a related embodiment, the ethod of diagnosing a disease caninclude reviewing or analyzing data relating to the presence of abiomolecule target or a biomolecule associated therewith and providing aconclusion to a patient, a health care provider or a health caremanager, the conclusion being based on the review or analysis of dataregarding a disease diagnosis. Reviewing or analyzing such data can befacilitated using a computer or other digital device and a network asdescribed herein. It is envisioned that information relating to suchdata can be transmitted over the network.

In practicing the methods of the present invention, many conventionaltechniques in molecular biology are optionally utilized. Thesetechniques are well known and are explained in, for example, Ausubel etal. (Eds.) Current Protocols in Molecular Biology, Volumes I, II, andIII, (1997), Ausubel et al. (Eds.), Short Protocols in MolecularBiology: A Compendium of Methods from Current Protocols in MolecularBiology, 5^(th) Ed., John Wiley & Sons, Inc. (2002), Sambrook et al.,Molecular Cloning: A Laboratory Manual, 3rd Ed., Cold Spring HarborLaboratory Press (2000), and Innis et al. (Eds.) PCR Protocols: A Guideto Methods and Applications, Elsevier Science & Technology Books (1990),all of which are incorporated herein by reference.

FIG. 22 is a block diagram showing a representative example logic devicethrough which reviewing or analyzing data relating to the presentinvention can be achieved. Such data can be in relation to a disease,disorder or condition in a subject. FIG. 22 shows a computer system (ordigital device) 800 connected to an apparatus 820 for use with themultichromophore or multichromophore complexes 824 to, for example,produce a result. The computer system 800 may be understood as a logicalapparatus that can read instructions from media 811 and/or network port805, which can optionally be connected to server 809 having fixed media812. The system shown in FIG. 22 includes CPU 801, disk drives 803,optional input devices such as keyboard 815 and/or mouse 816 andoptional monitor 807. Data communication can be achieved through theindicated communication medium to a server 809 at a local or a remotelocation. The communication medium can include any means of transmittingand/or receiving data. For example, the communication medium can be anetwork connection, a wireless connection or an internet connection. Itis envisioned that data relating to the present invention can betransmitted over such networks or connections.

In one embodiment, a computer-readable medium includes a medium suitablefor transmission of a result of an analysis of a biological sample. Themedium can include a result regarding a disease condition or state of asubject, wherein such a result is derived using the methods describedherein.

Kits

Kits comprising reagents useful for performing described methods arealso provided.

In some embodiments, a kit comprises reagents including multichromophoreor multichromophore complexes, bioconjugates, for example, antibodies,and other components as described herein.

The kit may optionally contain one or more of the following: one or morelabels that can)e incorporated into multichromophore or multichromophorecomplexes, and one or more substrates which may or may not contain anarray, etc.

The components of a kit can be retained by a housing. Instructions forusing the kit to perform a described method can be provided with thehousing, and can be provided in any fixed medium. The instructions maybe located inside the housing; or outside the housing, and may beprinted on the interior or exterior of any surface forming the housingthat renders the instructions legible. A kit may be in multiplex formfor detection of one or more different target biomolecules orbiomolecules associated therewith.

As described herein and shown in FIG. 23, in certain embodiments a kit903 can include a container or housing 902 for housing variouscomponents. As shown in FIG. 23, and described herein, in one embodimenta kit 903 comprising one or more multichromophore or multichromophorecomplexes reagents 905, and optionally a substrate 900 is provided. Asshown in FIG. 23, and described herein, the kit 903 can optionallyinclude instructions 901. Other embodiments of the kit 903 areenvisioned wherein the components include various additional featuresdescribed herein.

EXAMPLES Example 1

General protocol for the sandwich ELISA method with polymer-dyeconjugated antibody:

1. Bind the unlabeled antibody to the bottom of each well by addingapproximately 50 μL of antibody solution to each well (20 μg/mL in PBS)in a 96 wells polyvinylchloride (PVC) microliter plate. PVC will bindapproximately 100 ng/well (300 ng/cm2). The amount of antibody used willdepend on the individual assay.

2. Incubate the plate overnight at 4° C. to allow complete binding.

3. Wash the wells twice with PBS.

4. The remaining sites for protein binding on the microtiter plate mustbe saturated by incubating with blocking buffer. Fill the wells to thetop with 3% BSA/PBS with 0.02% sodium azide. Incubate for 2 hrs. toovernight in a humid atmosphere at room temperature.

5. Wash wells twice with PBS.

6. Add 50 μL of the antigen (or sample) solution to the wells (theantigen solution should be titrated). All dilutions should be done inthe blocking buffer (3% BSA/PBS). Incubate for at least 2 hrs. at roomtemperature in a humid atmosphere.

7. Wash the plate four times with PBS.

8. Add access amount of the either polymer-dye-second antibodyconjugates (Example A or C) or biotin-labeled antibody.

9. Incubate for 2 hrs, or more at room temperature in a humidatmosphere.

10. Wash with several changes of PBS,

11. When the biotin-labeled antibody is used in Step 8, addstreptavidin-polymer-dye conjugate (Example B or D, in PBS containing 1M NaCl) and incubate for 2 hrs. or more at room temperature in a humidatmosphere

12. Measure optical densities at target wavelengths on an ELISA platereader.

For quantitative results, compare signal of unknown samples againstthose of a standard curve. Standards must be run with each assay toensure accuracy.

In the ELISA assays, the primary antibody nolecules are bound on theside and bottom of the wells in a microliter plate. When the samplecontaining the target molecules is added into the well, the immobilizedprimary antibody will only capture those targets and the rest of thecomponents in sample will be washed away. Comparing to the commonly usedfluorescence-labeled antibody, the described polymer-dye-secondaryantibody conjugates may emit a much stronger signal (10-100 fold) thanthe regular setup due to their higher light harvesting capability andtheir within-the-same-molecule design for better energy transferefficiency. These advantages can also be translated into an assay withhigher sensitivity. When further comparing the polymer-dye-secondaryantibody conjugates with the other secondary antibody equipped with asignal amplification functionality (e.g., horseradish peroxidase labeledantibody), the polymer-dye-secondary antibody conjugates can provide aone-step process (without additional enzymatic substrate) to achieve thepurpose of signal amplification. The cost effectiveness (in both of timeand material) of the described conjugates is also anticipated to have abetter market acceptance.

Example 2

General protocol for microarray labeling with polymer-dye conjugatedantibody:

1. Prepare total RNA or mRNA.

2. Use T7-oligo(dT) primer to perform one-cycle or two-cycle cDNAsynthesis.

3. Cleanup of double stranded cDNA.

4. Use IVT (in vitro transcription) amplification kit to incorporatebiotin-labeled ribonucleotide into cRNA.

5. Fragmentation of cRNA.

6. Hybridize cRNA fragments on chip.

7. Wash off residual cRNA and stain the chip with strep in-polymer-dyeconjugate (Example B or D)

8. Wash off residual reagents on chip.

9. Scan microarray.

In the regular practice of microarray methodology, an integration ofbiotin-labeled nucleotides into the cRNA sequences is the means ofsequestering the septavidin phycoerythrin conjugate and biotinylatedanti-streptavidin antibody for amplified signal reporting. Due to themanufacture complexity of streptavidin phycoerythrin conjugate, thebatch-to-batch variation is significant. Therefore, thestreptavidin-polymer-dye conjugate can be a very good alternative toreplace streptavidin phycoerythrin conjugate. Furthermore, priorpublications have demonstrated that MULTICHROMOPHORES can amplify thefluorescence signals up to 75-fold through its light harvesting andenergy transfer functionalities. It is reasonable to anticipate that thestreptavidin-polymer-dye conjugate may perform equivalently or betterthan phycoerythrin.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the an thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

Example 3

Synthesis of cationic conjugated polymer with an amine functional group,CA0011:

1-(4′-Phthalimidobutoxy)-3,5-dibromobenzene: 3,5-dibromophenol (970 mg,3.85 mmol) was recrystallized from hexanes. After removal of solvent,N-(4-bromobutyl)phthalimide (1.38 g, 4.89 mmol), K₂CO₃ (1.88 g, 13.6mmol), 18-crown-6 (53 mg, 0.20 mmol), and acetone (20 mL) were added.This was refluxed for 1 hour, and then poured into 100 mL of water. Theaqueous layer was extracted with dichloromethane (4×30 mL). The organiclayers were combined, washed with water, saturated NaHCO₃, and brine,then dried over MgSO₄ and filtered. Removal of solvent yielded a whitesolid, which was purified by column chromatography (4:1 hexanes:CH₂Cl₂)followed by recrystallization in hexanes to yield colorless needles (650mg, 87%). ¹H NMR (CDCl₃): 7.860 (m, 2H); 7.733 (m, 2H); 7.220 (t, J=1.6Hz, 1H); 6.964 (d, J=2.0 Hz, 2H); 3.962 (t, J=6.0 Hz, 2H); 3.770 (t,J=6.6 Hz, 2H);1.846 (m, 4H).

Poly[(2,7-{9,9-bis(6′-bromohexyl)}fluorene-co-alt-1,4-{2,5-difluoro}phenylene)-co-(2,7-{9,9-bis(6′-bromohexyl)}fluorene-co-alt-3,5-1-{4′-phthalimidobutoxy)phenylene)]:A solution of2,7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-9,9-bis(6′-bromohexyl)fluorene(1.001 g, 1.34 mmol), 1,4-dibromo-2,5-difluorobenzene (346.6 mg, 1.274mmol), 1-(4′-phthalimidobutoxy)-3,5-dibromobenzene (30.8 mg, 0.068mmol), potassium carbonate (2.15 g, 15.5 mmol), andtetrakis(triphenylphosphine)palladium (0) (37.2 mg, 0.032 mmol)) in THF(45 mL) and water (15 mL) in a 100 mL round-bottomed flask equipped witha water jacketed reflux condenser was degassed via four freeze-pump-thawcycles, with argon being introduced after the third and fourth round ofdegassing. The solution was then heated to reflux for 48 hours under anargon atmosphere. After cooling, the solution was added dropwise to 40mL of stirring methanol to precipitate the polymer, which was collectedby centrifugation. This was followed by decanting and washing withmethanol (twice) to remove low molecular weight fractions, yielding apale yellow, fluffy powder (500 mg, 62%). 1H NMR (CD2Cl2): 7.912-7.419(m, 8H); 3.322 (t, J=7.4 Hz, 4H); 2.120 (br s, 4H); 1.693 (t, J=7.0 Hz,4H); 1.237 (br s, 4H); 1.153 (br s, 4H); 0.788 (br s, 4H), Mn 17K, PDI2.1.

Poly[(2,7-{9,9-bis(6′-(N,N,N-trimethylammoniumbromide)hexyl)}fluorene-co-alt-1,4-{2,5-difluoro}phenylene)-co-(2,7-{9,9-bis(6′-(N,N,N-trimethylammoniumbromide)hexyl)}fluorene-co-alt-3,5-1-{4′-phthalimidobutoxy)phenylene)]:Trimethylamine (1 mL) was condensed into a solution ofpoly[(2,7-{9,9-bis(6′-bromohexyl)}fluorene-co-alt-1,4-{2,5-difluoro}phenylene)-co-(2,7-{9,9-bis(6′-bromohexyl)}fluorene-co-alt-3,5-1-{4′-phthatimidobutoxy)phenylene)](130 mg, 0.215 mmol) in THF (10 mL) under reduced pressure. Thissolution was stirred for 24 h, at which point the polymer precipitatedfrom solution. Methanol was added (50 mL) to solubilize the polymer,then another 1 mL, of trimethylamine was condensed into the reactionflask under reduced pressure. This was stirred an additional 24 hours,then all solvents and excess trimethylamine removed under reducedpressure to give a pale yellow film (140 mg, 90%). 1H NMR (D2O):7.871-7.423 (m, 8H); 3.148 (m, 4H); 2.970 (br s, 18H); 2.116 (br s, 4H);1.525 (br s, 4H); 1.119 (br s, 8H); 0.681 (br s, 4H).

CA001,poly[(2,7-{9,9-bis(6′-(N,N,N-trimethylammoniumbromide)hexyl)}fluorene-co-alt-1,4-{1,2,5-difluoro}phenylene)-co-(2,7-{9,9-bis(6′-(N,N,N-trimethylammoniumbromide)hexyl)}fluorene-co-alt3,5-1-{4′-aminobutoxy)phenylene)]:A solution of hydrazine monohydrate (73.1 mg, 1.46 mmol),poly[(2,7-{9,9-bis(6′-(N,N,N-trimethylammoniumbromide)hexyl)}fluorene-co-alt-1,4-{2,5-difluoro}phenylene)-co-(2,7-{9,9-bis(6′-(N,N,N-trimethylammoniumbromide)hexyl)}fluorene-co-alt-3,5-1-{4′-phthalimidobutoxy)phenylene)](100 mg, 0.137 mmol) in methanol (10 mL) were refluxed for 5 hours.After cooling to room temperature, 0.9 mL 1 M HCl were then added to thesolution, which was then refluxed for an additional 2 hours. Theresulting solution was dialyzed against 50% methanol in water, thenevaporated to dryness.

A method was determined for evaluating the incorporation of thefunctionalized monomers into the final polymer structures. The aminefunctional groups were protected as phthalimide during thepolymerization reaction to prevent catalyst contamination. Thisprotecting group has a unique signature in infrared (IR) spectroscopy,shown as a solid line in FIG. 24. The peaks indicated correspond tounique C═O peaks only present for the phthalimide protecting group. Postdeprotection of the phthalimide group (yielding a free amine) gives aCA001 IR signature that lacks the phthalimide's signature C═O peaks(dashed line, FIG. 24), and indicates an active amine available forconjugation.

The optical spectra of CA001 are shown in FIG. 25, where the solid lineindicates the absorption and the dotted line indicates the emissionspectra.

Example 4

Synthesis of cationic conjugated polymer-FAM conjugate, CA001-FAM:

The deprotected polymer CA001 (having a free amine) was reacted with asuccimidyl ester FAM, 5(6)FAM-SE (invitrogen, #C1311), adapted fromprotocols available at www.invitrogen.com (last visited OCT. 4, 2007).As a negative control, the same polymer was incubated with fluorescein(no reactive group) under the same reaction conditions. The protocol forthis procedure follows.

Conjugation of NHS-FAM to CA001

Purpose:

To biotinylate CA001 with NHS-FAM and demonstrate FRET tocovalently-bound dye.

Materials:

Fluorometer, with UV-transparent cuvettes

UV-VIS instrument

Purified CA001

NHS-FAM (Invitrogen #C-1311)

0.5 M NEt3 (1:14 dilution of stock (7.2M) NEt3)

MC30 filters

Procedure:

1. Set up reactions using a 10-fold XS of NH-FAM to amine-polymer. Use50 ug polymer and 8.0 ug NHS-FAM per 10 uL rxn:

NHS-FAM, Fluorescein, 8.0 mg/mL 8.0 mg/mL Sample uL Rxn 90% M1T CA0010.25M NEt3 DMSO DMSO CA001 + NHS-FAM: 10 (8 − x) uL x uL 1.0 uL 1.0 uL —CA001 − NHS-FAM: 10 1.0 — 1.0 uL

2. When all but dye have been combined, dissolve 1-2 mgs of dye in DMSOat 30 uL anhydrous DMSO/mg dye. Use NHS-reagents without delay afterdissolving.

3. Incubate @25C on heat block for 30 min.

4. Dilute in 90M1T (10 uL in 400 ul)

5. Desalt by MC30, 2×

6. Assay for concentration by UV/Vis

Resulting fluorescence spectra for each reaction product are shown inFIG. 26. The fluorescence arising from the positive control is shown asa solid line. When the polymer is excited, FRET to the acceptor dye ;nowcovalently bound to the polymer) occurs, resulting in intensefluorescein emission. The fluorescence arising from the negative controlis shown as a dotted line. Because the fluorescein for the negativecontrol cannot bind the polymer, when the polymer is excited, FRET doesnot occur and only polymer emission is observed. These data indicatethat the amine on CA001 is available for conjugation.

Example 5

Synthesis of a biotinylated conjugated polymer, biotinyl-CA001:

The amine functionality on CA001 was converted to a biotin functionalityusing an NHS-biotin linker available from Pierce (#20217). The protocolfor this procedure was modified from the Pierce protocol, found on thePierce website, ww.piercenet.com (last visited Sep. 23, 2007). Theprotocol for this procedure analogously follows that noted in Example12.

Example 6

Procedure for the amplification of signal by blotinyl-CA001, Avidin DN,and biotinyl-fluorescein:

Purpose:

To demonstrate fluorescent signal amplification via FRET, usingbiotinyl-CA001, Avidin DN, and biotinyl-fluorescein

Materials:

NanoDrop fluorometer

Perkin-Elmer fluorometer, model PE-LS55

UV-transparent plastic 1-ml cuvettes

pipeters+tips

biotinyl-CA001 (BCA)

CA001 (CA)

biotinyl-fluorescein (BFL)

Avidin DN (ADN)

TBS

Procedure:

1 In an eppendorf tube, combine reagents as listed in table below. Besure add ADN last and to mix together other reagents prior to additionof ADN. Dilute combinations 100-fold prior to measurement on afluorometer, either 1 uL in 100uL for the NanoDrop, or 10 ul in 1 mLcuvette for a benchtop fluorometer.

2. Directly excite fluorescein at 488 nm as well as indirectly via FRETby exciting the polymer at 380 nm.

3. Collect data on peak heights at relevant wavelengths. Subtractbackground from peak heights, including these sources

3a) Buffer alone control

3b) Polymer peak tail (˜5%) from FRET to fluorescein peaks

B-FL CA BCA ADN Exc. 415 nm 533 nm 533 nm TBS 5 uM 5 uM 5 uM 5 uM (nm)pk. ht. pk. ht. pk. ht. TBS only 20 uL 488 — 380 BFL only 20 uL 1 uL 488— CA only 20 uL 1 uL 380 BFL + CA + ADN, 20 uL 1 uL 1 uL 1 uL 488 1:1:1380 BFL + BCA + ADN, 20 uL 1 uL 1 uL 1 uL 488 1:1:1 380 BFL + CA + ADN,20 uL 1 uL 2 uL 1 uL 488 1:2:1 380 BFL + BCA + ADN, 20 uL 1 uL 2 uL 1 uL488 1:2:1 380

Example 7

Analysis of amplification of signal by Biotinyl-CA001, Avidin DN, andBiotinyl-fluorescein:

FIG. 27A shows the biotinylation of CA001. The amine polymer CA001(precursor to the biotinyl polymer) should not bind avidin, and is usedas the negative control polymer. FIG. 27B depicts the assayschematically. The biotinylated dye and polymer are brought togetherspecifically by biotin-avidin binding. The negative control polymer(amine polymer CA001, noted as CA) should not bind the avidin. FIG. 27Cshows the fluorescence spectra resulting from the assay followed in theabove protocol (Example 6). The dotted line shows the fluorescencespectra upon excitation at 380 nm of nonspecific polymer (CA) insolution with Avidin DN (AvDN) and biotinylated fluorescein (B-Fl). Onlypolymer emission, centered at 420 nm, is observed. The solid line showsthe fluorescence spectra upon excitation at 380 nm of hiotinylatedpolymer (BCA) in solution with Avidin DN (AvDN) and biotinylatedfluorescein (B-FI). Strong energy transfer is observed, resulting inadditional emission arising from the fluorescein, centered at 530 nm.FRET occurs only for biotinyl-CA001, indicating that the donorbiotinyl-CA001 and acceptor fluorescein are brought into close proximityvia biotin-avidin binding. This corroborates the biotinylation CA001following the Pierce procedure (Example 5). Direct excitation of the dyeat 488 nm is shown as a dashed line. Comparison of the dashed line withthe solid line reveals 19-fold amplification of the dye when excitedindirectly (via FRET) versus directly.

Example 8

Synthesis of cationic conjugated polymer precursor with a carboxylatefunctional group, CC001:

Poly[(2,7-{9,9-bis(6′-bromohexyl)}fluorene-co-alt-1,4-{2,5-difluoro}phenylene)-co-(2,7-{9,9-bis(6′-bromohexyl)}fluorene-co-alt-3,5-1-{7′-ethylesterheptoxy)phenylene)]:A solution of2,7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-9,9-bis(6′-bromohexypfluorene(500 mg, 0.670 mmol), 1,4-dibromo-2,5-difluorobenzene (173.2 mg, 0.637mmol), 1-(7′-ethylesterheptoxy)-3,5-dibromobenzene (13.6 mg, 0.033mmol), potassium carbonate (1.12 g, 8.12 mmol), andtetrakis(triphenylphosphine)palladium (0) (21 mg, 0.018 mmol) inTHF (15mL) and water (5 mL) in a 50 mL round-bottomed flask equipped with awater-jacketed reflux condenser was degassed via four freeze-pump-thawcycles, with argon being introduced after the third and fourth round ofdegassing. The solution was then heated to reflux for 48 hours under anargon atmosphere. After cooling, the solution was added dropwise to 40mL of stirring methanol to precipitate the polymer, which was collectedby centrifugation. This was followed by decanting and washing withmethanol (twice) to remove low molecular weight fractions, yielding apale yellow, fluffy powder. 1H NMR (CD2Cl2): 7.887-7.406 (m, 8H); 3.322(t, J=6.6 Hz, 4H); 2.080 (br s, 4H); 1.710 (t, J=7.0 Hz, 4H); 1.269 (brs, 4H); 1.158 (br s, 4H): 0.799 (br s. 4H). Mn 39.5K, PDI 2.1.

IR spectroscopy was used to evaluate the incorporation of thefunctionalized monomers into the final polymer structures. Thecarboxylate functional groups were protected as esters during thepolymerization reaction to prevent catalyst contamination. Thisprotecting group has a unique signature in infrared (IR) spectroscopy,as shown in FIG. 28. The peaks shown correspond to unique C═O peaks onlypresent for the carboxylate protecting group. The dashed line in FIG. 28corresponds to the IR spectra of the monomer, whereas the solid linecorresponds to the IR spectra of the polymer. In both cases, acarboxylate peak is observed, indicating incorporation of the functionalmonomer.

Example 9

Synthesis of an anionic conjugated polymer with an amine functionalgroup, AA003:

Poly[(2,7-{9,9-bis(4′-(sodiumsulfonate)butyl)}fluorene-co-alt-1,4-{2,5-difluoro}phenylene)-co-(2,7-{9,9-bis(4′-(sodiumsulfonate)butyl)}fluorene-co-alt-3,5-1-{4′-phthalimidobutoxy)phenylene)]:A solution of 2,7-dibromo-9,9-bis(4′-(sodiumsulfonate)butyl)fluorene(129.5 mg, 0.202 mmol), 1,4-diboronic acid (37.4 mg, 0.225 mmol),1-(4′-phthalimidobutoxy)-3,5-dibromobenzene (10.4 mg, 0.023 mmol),potassium carbonate (366 mg, 2.65 mmol), andtetrakis(triphenylphosphine)palladium (0) (8.8 mg, 0.008 mmol) in DMF(20 mL) and water (20 mL) in a 100 mL round-bottomed flask equipped witha water-jacketed reflux condenser was degassed via four freeze-pump-thawcycles, with argon being introduced after the third and fourth round ofdegassing. The solution was then heated to reflux for 48 hours under anargon atmosphere. Over the course of the reaction, a black precipitateformed. After cooling, the solution was removed and the precipitate waswashed with acetone to give a brown powder, 1H NMR (DMSO): 7.910-7.597(m, 10H); 2.206 (br s, 8H); 1.400 (br s, 4H); 0.668 (br s, 4H). IR: 1696cm-1, indicating a protected amine (phthalimide).

AA003,Poly[(2,7-{9,9-bis(4′-(sodiumsulfonate)butyl)}fluorene-co-alt-1,4-}2,5-difluoro}phenylene)-co-(2,7-{9,9-bis(4′-(sodiumsulfonate)butyl)}fluorene-co-alt--3,5-1-{4′-aminobutoxy)phenylene)]:A solution of hydrazine monohydrate (29.9 mg, 0.598 mmol) andpoly[(2,7-{9,9-bis(4′-(sodiumsulfonate)butypl)}fluorene-co-alt-1,4-{2,5-difluoro}phenylene)-co-(2,7-{9,9-bis(4′-(sodiumsulfonate)butyl)}fluorene-co-alt-3,5-1-{4′-aminobutoxy)phenylene)] (45 mg, 0.081 mmol) in 50%methanol/water (5 mL) were refluxed for 5 hours. After cooling to roomtemperature, the pH of the solution was adjusted to 3 with 1M HCl, thenrefluxed for an additional 2 hours. After cooling and transfer to a 15mL Falcon tube, AA003 was purified by the following protocol.

Purifying Deprotected AA003

Purpose:

To enrich for amine-activated anionic polymer

Materials:

UV-transparent plastic 1-mL cuvettes

centrifuge

UV-Vis spectrophotometer

pipeters+tips

NaOH, 1.0 M, 0.1 mL:

NaOH, 10M 10 uL H2O 90 uL

Crude AA003

90% MeOH, 1% T20 (90MIT, 50 mL)

MC30 filters

Procedure:

1. Sort out fractions:

1a) If there is a film lining the inside of the tube (precipitatedpolymer), pour off the sup mate, into a new tube, Using a pipet tip,remove all the supernate completely, and set it aside.

1b) Process the film (ppt) lining the tube:

-   -   i. Add 0.5 mL water and check pH by litmus. If necessary,        neutralize to pH 7-8 by adding NaOH (1-5 uL amounts of 0.1M NaOH        at first, then of 1.0 M NaOH if more practical), mix, pellet,        check on litmus. Go through several add-mix-pellet-check pH test        cycles until pH 8 is maintained.    -   ii. Remove water extract, and place in a 1.5 mL eppi.    -   iii. Spin @ 1.4krcf, 2′. Save sup. and pellet.    -   iv. Lyophilize pellet.

1c) Process the supernate from step 1a):

-   -   i. Sample 0.5mL of supernate (usually a suspension) and place in        a 1.5 mL eppi.    -   ii. Test pH by litmus and record pH.    -   iii. Neutralize to pH 7-8 as in step 1b1 above.    -   iv. Centrifuge @ 14krcf, 2′.    -   v. Separate sup. from pellet, keeping both.    -   vi. Lyophilize pellet,    -   vii. Re-suspend pellet in minimal amt. of anhydrous DMSO.

viii. Perform UV/Vis spec. in water, record absorbance and calculateconcentration of all fractions, using valid Ext. Coef.

2. Desalt by MC30.

The optical spectra of AA003 are shown in FIG. 29, where the solid lineindicates the absorption and the dashed line indicates the emissionspectra. The deprotected polymer AA003 (having a free amine) was reactedwith a succimidyl ester FAM, 5(6)FAM-SE (Invitrogen, #C1311), adaptedfrom protocols available at www.invitrogen.com (last visited Oct. 4,2007). As a negative control, the same polymer was incubated withfluorescein (no reactive group) under the same reaction conditions. Theprotocol for this procedure analogously follows that of Example 4.

Example 10

Synthesis of anionic conjugated polymer with a maleimide functionalgroup AA003-M01:

The amine functionality on a multichromophore can be converted to otherfunctionalities with the use of a dual-functional linker, such as GMBS.This strategy was taken to convert AA003 to AA003-M01. The protocol forthis procedure was modified from the Pierce protocol, found on thePierce website, www.pierce.net,com (last visited Sep. 23, 2007). Theprotocol used can be found below.

Conjugation of GMBS to AA003

Purpose:

To functionalize AM003 with GMBS to give a maleimide moiety

Materials:

Centrifuge

UV-Vis spectrophotometer

UV-transparent, cuvettes

pipeters+tips

AA003

GMBS (Pierce #22309)

90% MeOH, 1% T20 (90M1T, 50 mL)

DMSO

NEt3

MC30 filters

Procedure:

1. To functionalize AA003 with Maleimide, here are useful amounts touse:

1a) For 40× XS, use 0.3 mM polymer and 12 mM GMBS. This means 3.0 nmolpolymer/10 uL rxn as follows:

Sample Rxn. Vol. 90% M1T DMSO AA003 0.5M NEt3 GMBS, 120 mM AA003 + G 10uL 5.7 uL — 2.3 uL 1.0 uL 1.0 uL AA003 − G 10 5.7 1.0 uL 2.3 uL 1.0 —

2. When all reagents but linker have been combined:

2a) Dissolve GMBS in DMSO (Use GMBS without delay after dissolving):

-   -   i. 120 mM GMBS=3.4 mg/0.1 mL DMSO, or 30 uL/mg

2b) Incubate @ 25C on heat block for 30 min. Check for clarity.

3. Remove XS GMBS by MC30:

3a) Before applying to MC cup, dilute DMSO to <5%

3b) Separately combine each reaction w/400 uL 90M1T

3c) Apply to cup

3d) Spin 10 mins @ 14k ref

3e) Determine if more time spinning is necessary by estimating volume ofretentate

3f) Discard filtrate

3g) Add 400 uL more 90M1T and repeat spin

3h) If rerentate looks nearly dry, add 20 uL, 90M1T and swirl a bit incup

3i) Invert and spin to collect retenate

3j) Measure final retentate volumes=_uL

3k) Determine concentration by UV-vis, then adjust final concentrationto 25 uM in 0.5× 90M1T.

Example 11

Synthesis of anionic conjugated polymer fluorescein conjugateAA003-M01.-FI:

The maleimide functional group on AA003-M01 was tested for thiolreactivity by reacting with SAMSA-fluorescein (Invitrogen) usingprotocols adapted from www.invitrogencom (last visited Oct. 4, 2007).AA003 was used as a negative control. The modified protocol can be foundbelow.

SAMSA assay for Maleimide

Purpose: To demonstrate AA003-M01 has an active maleimide moiety using10× XS SAMSA-fluorescein to AA003-M01

Materials:

Centrifuge

UV-transparent, 1-mL cuvettes

Fluorometer. model PE-L555

pipeters+tips

potassium phos, 0.5M, pH 7.0, 0.2 mL:

K2HPO4, 1M  62 uL KH2PO4, 1M  39 uL H2O 100 uL

HCl. 6 M. 0.2 mL:

H2O 0.1 mL HCl, 12M 0.1 mL

NaOH, 0.1 M. 1 mL:

NaOH, 10M  10 uL H2O 990 uL

AA003

AA003-M01

SAMSA-fluorescein (invitrog,en, product A685)

MC30 filters

Procedure:

1. Prepare 1.0 mM deprotected SAMSA:

1a) Dissolve 1.0 mg SAMSA/95 uL. of 0.1. M NaOH (20 MM SAMSA)

1b) Incubate at RT for 15 mins to remove acetyl protecting group

1c) Neutralize with 6 M HCl: 1.4 uL/mg SAMSA (20 mM SAMSA)

1d) Buffer with 20 uL 0.5M sodium phos, pH 7/mg SAMSA (16 mM SAMSA)

1e) Dilute 16-Fold to 1.0 mM SAMSA with water.

2. Setup rxns by adding:

2a) For AA003-M01: 10 uL of 1.0 mM deprotected SAMSA to 10 uL, of 25 uMAA003+GMBS (3.k+G from protocol in Example 10)

2b) For AA003: 10 uL of 1.0 mM deprotected SAMSA to 10 uL of 25 uMAA003-GMBS (3.-G from protocol in Example 10)

3. Incubate on heat block @ 25 C, 30 mins

4. Remove XS SAMSA by MC30:

4a) Before applying to MC cup, dilute DMSO to <5%

4b) Separately combine each rxn w/400 uL 90M1T

4c) Apply to cup

4d) Spin 10 mins @ 14k ref

4e) Determine if more time spinning is necessary by est vol of retentate

4f) Discard filtrate

4g) Add 400 uL more 90M1T and repeat spin

4h) If retentate looks nearly dry, add 20 uL, 90M1T and swirl a bit incup

4i) Invert and spin to collect retentate

5. Analyze fluorescence @ 488 and 380 excitation of both AA003-M01 andAA003 samples from step 4i.

FIG. 30C shows the results from this assay. AA003-M01 (FIG. 30B noted asMaleimide-functionalized polymer) and a negative control polymer (nomaleimide, AA003 FIG. 30A noted as Negative control polymer) werereacted with a thiolated fluorescein (SAMSA-fluorescein) according theabove procedure. The maleimide-functionalized polymer AA003-M01 reactswith the thiolated fluorescein, and becomes covalently bound to thefluorescein, ensuring a fixed distance between the donor polymer and theacceptor dye. Thus, excitation of the polymer results in FRET to theacceptor dye, and intense dye emission is observed (solid line, FIG.30C). The negative control does not covalently bind fluorescein, andwhen the polymer is excited, only polymer emission is observed (dottedline, FIG. 30C).

Example 12

Synthesis of a biotinylated anionic conjugated polymer. Biotinyl-AA003:

The amine functionality on AA003 was converted to a biotin using anNHS-biotin linker available from Pierce (#20217). The protocol for thisprocedure was modified from the Pierce protocol, found on the Piercewebsite, www.piercenet.com (last visited Sep. 23, 2007). The protocolused can be found below.

Procedure for the Conjugation of NHS-Biotin to AA003

Purpose:

To biotinylate AA003 with NHS-Biotin.

Materials:

Fluorometer, with UV-transparent cuvettes

UV-VIS instrument

Purified AA003 (AA3)

NHS-biotin (Pierce #20217)

0.5 M NEt3 (1:14 dilution of stock (7.2M) NEt3)

DMSO

MC30 filters

Procedure:

1. Set up reactions, 0.5 mM polymer and 20 nM NHS-biotin:

AA3, NHS- uL 90% 5.0 nmol/ 0.5M biotin, Sample Rxn M1T DMSO 10 ul NEt3200 mM PC: CA1 + Biot 10 6.8 uL — 1.2 uL 1.0 uL 1.0 uL NC: CA1 − Biot 106.8 1.0 uL 1.2 uL 1.0 — AA3 + B 40 0 — 38 uL 4.0 4.0 uL AA3 + B 10 0 1.09.6 uL 1.0 — 2b3SUP + B 35 0 — 34 uL 3.5 3.5 uL 2b3SUP − B 10 0 1.0 9.8uL 1.0 — 2b3PEL + B 70 20 — 36 uL 7.0 7.0 uL 2b3PEL − B 10 2.9 1.0 5.1uL 1.0 —

2. When all but biotin has been combined, dissolve 1-2 mgs of NHS-biotinin DMSO at 15 uL anhydrous DMSO/mg NHS-biotin (or 1.7 mg/0.025 mL; 200mM). Use NHS-reagents without delay after dissolving.

3. Incubate @ 25C on heat block for 30 min.

4. Dilute 1:100 in 90M1T (1 uL in 100, or 4 in 400 uL)

5. Desalt by MC30, 2×

5. Assay for concentration by UN/Vis

Example 13

Procedure for amplification of signal by Bio -AA003 and AvidinD-fluorescein:

Purpose:

To demonstrate specific fluorescent signal via FRET using biotinyl-AA003and Avidin D-Fluorescein.

Materials:

Fluorometer (NanoDrop or Perkin-Elmer PE-LS55)

UV-transparent plastic 1-mL cuvettes

pipeters+tips

biotinyl-AA003 (BAA)

AA003 (AA)

Avidin D-Fluorescein (A-Fl)

TBS

Procedure:

1. In an eppendorf tube, combine reagents as listed in table below.Incubate for five minutes, then dilute combinations 100-fold prior tomeasurement on a fluorometer, either 1 uL in 100 uL for the NanoDrop, or10 uL in 1 mL cuvette for a benchtop fluorometer.

2. Directly excite A-Fl at 488 nm as well as indirectly via FRET byexciting the polymer at 380 nm.

3. Collect data on peak heights at relevant wavelengths. Subtractbackground from peak heights, including these sources

3a) Buffer alone control

3b) Polymer peak tail (-5%) from FRET to fluorescein peaks

A-Fl AA BAA Exc. 415 nm 566 nm 533 nm TBS 5 uM 5 uM 5 uM (nm) pk. ht.pk. ht. pk. ht. TBS only 20 uL 550 — 380 AA only 20 uL 3 uL 380 AA +A-Fl 6:1 20 uL 0.5 uL 3 uL 550 380 BAA only 20 uL 3 uL 380 BAA + A-Fl6:1 20 uL 0.5 uL 3 uL 3 uL 488 380

Example 14

Analysis of amplification of signal by Biotinyl-AA003 andfluorescein-labeled Avidin D:

This assay is described procedurally in Example 13. A scheme of thisassay is shown in FIG. 31B. Biotinyl-AA003 (see FIG. 31A noted asBiotinyl polymer) is incubated with fluorescein-labeled Avidin D. As anegative control, the amine polymer AA003 (see FIG. 31A noted asNegative control polymer) is incubated with fluorescein-labeled Avidin Din a similar manner. In each case, the polymer is excited, andfluorescein emission observed. For the negative control AA003, onlypolymer emission (centered at 420 nm) is observed, whereas for thebiotinyl-AA003, strong dye emission is observed.

Results from this assay are shown in FIGS. 32A-B. The biotinyl-AA003 wastested with Avidin D containing 4 dyes per avidin. Signals from thecontrol polymer and the dye alone were also recorded and are presentedin FIGS. 32A-B. FIG. 32A shows the resulting fluorescence spectra. Thedye signals at 523 nm from this data set are summarized in FIG. 32B.

These data indicate the fluorescein signal (at 523nm) is amplified inthe presence of the polymer (FIG. 32A, solid vs dashed spectra, left andbiotinyl-AA003 vs dye alone, right) and the signals observed were due tospecific polymer-Avidin D complexes (FIG. 32A, solid vs dotted spectra,left and biotinyl-polymer vs control, right). The amine polymer control(no biotin) was not able to bind the avidin and thus minimal energytransfer was observed (88% specificity). Specificity is defined as1-(control signallspecific dye signal). The right figure illustratesthe, difference in dye signal with and without polymer and between thepositive and negative control samples. The data presented in FIG. 32Bare corrected for signals arising from buffer and polymer tail. Thepolymer tail contribution at 523nm is 5% of the polymer peak height at419 nm.

Example 15

Amplification effects of varying Dye:Avidin D ratios:

Different ratios of polymer to dye were tested. This was evaluated usingAvidin D conjugates from Vector Laboratories which contained an averageof 0.8, 1.5 and 4 fluorescein dyes per avidin. As the number of dyes isincreased the ratio of extinction coefficients (absorbance) between thepolymer and dye is decreased. It was therefore predicted that for thisset of fluorescein-labeled Avidin D. the best amplification values wouldbe observed for the avidin conjugates containing the lowest number ofdyes. Polymer concentration was held constant at two equivalents ofpolymer per Avidin D.

The data shown in FIGS. 33A-B indicate a dependence on the ratio ofpolymer to dye as was expected. This ratio was varied by increasing thenumber of dyes per Avidin D at constant polymer and avidinconcentrations. The data indicate that as the ratio of dye:avidinincreases from 0.8 to 4 as the signal intensity of the dye increases(FIG. 33A) while the observed amplification drops (FIG. 33B). As thenumber of dyes increases, so does the direct dye signal due to thehigher absorbance and thus higher fluorescence (gray bars, FIG. 33A).

Example 16

Synthesis of anionic conjugated polymer with an amine functional group,AA002, capable of 405 nm excitation:

Poly[2,7-{9,9-bis(1-(2-(2-methoxyetboxy)ethoxy)ethoxy))))}-co-alt-(2,7-{9,9-bis(4′-(sodiumsulfonate)butyl)}fluorene-co-2,7-{9,9-bis(1-(2-(2-methoxyethoxy)ethoxy)ethoxy)))}-co-alt-3,5-1-{4′-phthalimidobutoxy)phenylene)]:A solution of2,7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-9,9-bis(1-(2-(2-methoxyethoxy)ethoxy)ethoxy)))fluorene(120.1 mg, 0.150 mmol),2,7-dibromo{9,9-bis(4′-(sodiumsulfonate)butyl))fluorene (91.3 mg, 0.143mmol), 1-(4′-phthalimidobutoxy)-3,5-dibromobenzene (3.7 mg, 0.0082mmol), potassium carbonate (234 mg, 1.7 mmol), andtetrakis(triphenylphosphine)palladium (0) (6.2 mg, 0.0054 mmol) in DMF(3.8 mL), THF (2.5 mL), and water (2.5 mL) in a 48 mL Schlenck tube wasdegassed via sparging with argon for 20 minutes. The solution was thenheated to 85° C. for 3 days under an argon atmosphere. The solution wasadded drop wise into stirring acetone to give a dark brown solid, Thesolid was then stirred with methanol, the filtrate collected, and thesolvent removed to yield a bright yellow solid. 1H NMR (DMSO):8.055-7.828 (m, 12H); 3.558-3.293 (m, 24H); 3.188 (m, 12H); 2.217-2.161(m, 8H); 1,417 (br s, 4H); 0.664 (br s, 4H). Bimodal, Mn 7.3K, PDI 1.02,and Mn 49K, PDI1.2.

This polymer was then deprotected and purified to give AA002, usingprocedures analogous to those noted in Example 9. The optical spectrafor AA002 are shown in FIG. 34, where the solid line indicates theabsorption and the dashed line indicates the emission spectra.

Example 17

Synthesis of a biotinylated conjugated polymer, Biotinyl-AA002:

The amine functionality on AA002 was converted to a biotin functionalityusing an NHS-biotin linker available from Pierce (#20217). The protocolfor this procedure was modified from the Pierce protocol, found on thePierce website, www.piercenet.com (last visited Sep. 23, 2007). Theprotocol for this procedure analogously follows that noted in Example12.

Example 18

Amplification effects of varying polymer:Avidin D ratios

Fluorescein-Labeled Avidin D, or Avidin D-FI (0.8 dyes per avidin), heldat a constant concentration, was incubated with a series of increasingbiotinyl-AA002 concentrations ranging from 0 to 8 equivalents. This isshown schematically in FIG. 35A for the first two equivalents ofbiotinyl-AA002. For each ratio, dye fluorescence was recorded for directand indirect excitation (via FRET). As the polymer to dye ratioincreased, signals arising from direct excitation remained fairlyconstant, whereas the signals arising from indirect excitationincreased, as shown in FIG. 35B a plot of fluorescein emission as afunction of the ratio of AA002 to Avidin D-Fl. A plateau of thisincrease in signal was reached at roughly four equivalents ofbiotinyl-AA002, consistent with the occupation of all the biotin bindingsites on the Avidin D.

These data are consistent with the specific binding of biotinyl-AA002 tofluorescein-labeled Avidin D. High signal amplification is observed,which plateaus at four equivalents of polymer, indicating the occupationof all available biotin binding sites.

Example 19

Electrostatic amplification of dye signals

As shown schematically in FIG. 36B, dye-labeled proteins (Cy3-labeledIgG and fluorescein-labeled BSA) were each independently incubated withcationic polymer FFP-2F (see FIG. 36A). Nonspecific electrostaticassociation occurred between the polymer and each dye-labeled protein.Each solution was excited at 380 nm, and the emission spectra collected.These spectra were compared with the emission spectra collected fromdirect excitation of the dye, as shown in FIG. 36C indicating 30-foldamplification of Cy3-labeled IgG and 25-fold amplification offluorescein-labeled BSA. The Cy3 labeled IgG used was ananti-digoxigenin antibody which is used to target digoxigenin labeledantibodies.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to these skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

1.-71. (canceled)
 72. A multichromophore-biomolecule conjugatecomprising: a sensor biomolecule covalently linked to a light harvestingmultichromophore via a bioconjugation site, wherein the sensorbiomolecule is selected from a protein, an antibody or a nucleic acid;wherein the light harvesting multichromophore is a conjugated polymerhaving a backbone of π-conjugated repeat units and comprises thestructure:

wherein: CP1 is present and is a polycyclic repeat unit; CP2 is presentand comprises an aromatic repeat unit; CP3 is present and comprises anaromatic repeat unit comprising a bioconjugation functional groupcapable of covalently linking to the sensor biomolecule or a dye; m is 1to 10,000; n is independently 1 to 10,000 and; and a, b and c areindependently 1 to 250;
 73. The multichromophore-biomolecule conjugateaccording to claim 72, wherein the π-conjugated repeat units arerandomly distributed along the polymer backbone.
 74. Themultichromophore-biomolecule conjugate according to claim 72, whereinCP1 comprises 1 to 5 aromatic rings.
 75. Themultichromophore-biomolecule conjugate according to claim 72, whereinCP1 comprises up to 5 fused and/or bridged rings.
 76. Themultichromophore-biomolecule conjugate according to claim 72, whereinCP1 comprises a biphenyl.
 77. The multichromophore-biomolecule conjugateaccording to claim 76, wherein the biphenyl is bridged.
 78. Themultichromophore-biomolecule conjugate according to claim 74, whereinCP1 comprises fluorene.
 79. The multichromophore-biomolecule conjugateaccording to claim 78, wherein CP1 comprises a substituted fluorene. 80.The multichromophore-biomolecule conjugate according to claim 72,wherein the sensor biomolecule is an antibody or streptavidin.
 81. Themultichromophore-biomolecule conjugate according to claim 80, whereinthe sensor biomolecule is an antibody.
 82. Themultichromophore-biomolecule conjugate according to claim 72, whereinthe multichromophore further comprises one or more additionalπ-conjugated repeat units.
 83. The multichromophore-biomoleculeconjugate according to claim 73, wherein CP1 comprises a substitutedfluorene.
 84. The multichromophore-biomolecule conjugate according toclaim 80, wherein CP1 comprises a substituted fluorene.
 85. Themultichromophore-biomolecule conjugate according to claim 82, whereinCP1 comprises a substituted fluorene.
 86. An aqueous compositioncomprising: a multichromophore-biomolecule conjugate comprising a sensorbiomolecule covalently linked to a light harvesting multichromophore viaa bioconjugation site, wherein the sensor biomolecule is selected from aprotein, an antibody or a nucleic acid; wherein the light harvestingmultichromophore is a conjugated polymer comprising a backbone ofπ-conjugated repeat units selected from: CP1 that is a polycyclic repeatunit; CP2 that is an aromatic repeat unit; and CP3 that is an aromaticrepeat unit a comprising a bioconjugation functional group capable ofcovalently linking to the biomolecule or a dye; wherein the π-conjugatedrepeat units are randomly distributed along the polymer backbone. 87.The aqueous composition according to claim 86, wherein CP1 comprises 1to 5 aromatic rings.
 88. The aqueous composition according to claim 87,wherein CP1 comprises up to 5 fused and/or bridged rings.
 89. Theaqueous composition according to claim 86, wherein CP1 comprises abiphenyl.
 90. The aqueous composition according to claim 89, wherein thebiphenyl is bridged.
 91. The aqueous composition according to claim 86,wherein CP1 comprises fluorene.
 92. The aqueous composition according toclaim 91, wherein CP1 comprises a substituted fluorene.
 93. The aqueouscomposition according to claim 86, wherein the sensor biomolecule is anantibody or streptavidin.
 94. The aqueous composition according to claim93, wherein the sensor biomolecule is an antibody.
 95. The aqueouscomposition according to claim 94, wherein the multichromophore furthercomprises one or more additional π-conjugated repeat units.
 96. Theaqueous composition according to claim 95, wherein CP1, CP2, and CP3 arepresent.
 97. The aqueous composition according to claim 86, wherein themultichromophore comprises the structure:

wherein: m is 1 to 10,000; n is independently 1 to 10,000 and; and a, band c are independently 1 to
 250. 98. The aqueous composition accordingto claim 93, wherein CP1 comprises a substituted fluorene.
 99. Theaqueous composition according to claim 95, wherein CP1 comprises asubstituted fluorene.
 100. The aqueous composition according to claim97, wherein CP1 comprises a substituted .